Protein-refolding material

ABSTRACT

The present invention provides a method for refolding protein produced, for example, by  Escherichia coli , that is inactive due to an as yet unformed higher order structure, or protein deactivated due to a change in conformation for some reason. The invention comprises a method, refolding kit, refolding agent, and molding that activate a native function or activity inherent to a protein through treatment with zeolite beta of protein produced, for example, by  Escherichia coli , that is inactive due to an as yet unformed higher order structure, or protein deactivated due to a change in conformation for some reason. The invention also comprises a method for producing an active protein that utilizes the same. As compared with conventional methods, the present invention can provide a novel method for activating protein function that is highly versatile and generalizable, that employs a simple and easy protocol, and that is inexpensive and enables repeated use of the function activator.

TECHNICAL FIELD

This invention relates to a method for activating the function ofinactive protein, and more particularly relates to a method foractivating the function of inactive protein by inducing the refolding ofa protein that is inactive because its higher order structure has yet tobe formed or protein that is deactivated due to a change in conformationfor some reason, thereby enabling activation or regeneration of a nativefunction inherent to the protein. This invention also relates to amethod for producing an active protein utilizing this method foractivating the function of inactive protein. The invention furtherrelates to reagents, substances, and materials used in protocols andsteps for activating the function of inactive protein, and moreparticularly relates to reagents, substances, and materials, a so-calledreagent kit, used in protocols and steps for inducing the refolding of aprotein that is inactive because its higher order structure has yet tobe formed or a protein that is deactivated due to a change inconformation for some reason, thereby enabling activation orregeneration of a native function inherent to the protein. The inventionalso relates to the activation of inactive protein using such a kit. Theinvention additionally relates to a function activator (refolding agent)and a function-activating molding for inactive protein and moreparticularly relates to certain types of devices, such as chips, thathave the capacity to induce the refolding of a protein that is inactivebecause its higher order structure has yet to be formed or a proteinthat is deactivated due to a change in conformation for some reason,thereby activating or regenerating a native function inherent to theprotein. The invention also relates to the activation of inactiveprotein, that is, the production or generation of active protein, usingsuch a device.

BACKGROUND ART

The actual processes and functions within living organisms are notcarried out by genes, but rather by the proteins produced from genes.The elucidation and analysis of protein structure and function is thusdirectly related to, for example, drugs and disease treatment, and istherefore of vital importance. As a consequence, efforts are activelyunderway with regard to the synthesis and production of various proteinsby a variety of methods, the investigation of the structures of theseproteins, and the elucidation of their action mechanism and role inliving organisms. As is well known at this time, protein function isdetermined not only by the sequence and chain length of the amino acidsthat make up a protein, but also by the ordered conformation (higherorder structure) assumed by the protein.

Protein synthesis is generally carried out using, for example, anEscherichia coli, insect cell, or mammalian cell expression system.Synthesis using insect or mammalian cells very often yields solubleprotein with a controlled higher order structure that assumes an orderlyconformation. The separation and purification processes in thesemethods, however, are very complex. Not only is recovery of the targetprotein time consuming and expensive, but very little protein isobtained. In contrast, protein synthesis by Escherichia coli involvessimple procedures, does not require a great deal of time to obtain thetarget protein, and also is not very expensive. As a consequence,methods that use Escherichia coli that incorporates the genetic coderesponsible for synthesis of a target protein have at the present timebecome the mainstream for protein synthesis and the correspondingproduction processes are also being established.

However, when protein from a higher organism such as humans issynthesized using an Escherichia coli expression system, the intendedprotein is in fact obtained in terms of the number of amino acids andtheir bonding sequence, i.e., in terms of the amino acid chain, but theobtained protein has a disordered conformation and an uncontrolledhigher order structure, that is, an insoluble protein is obtained, knownas an inclusion body, in which the amino acid chain is entangled. Thisinclusion body of insoluble protein naturally lacks the desiredfunctions and properties and lacks activity. As a result, an Escherichiacoli-based production process requires refolding of the inclusion body,that is, a process in which the inclusion body is unraveled andconverted into soluble protein with a modulated higher order structureand an orderly conformation.

This type of refolding is applied not only to protein produced byEscherichia coli, but is also applicable to the regeneration of proteinthat is deactivated by certain mechanisms, for example, the thermalhistory, and is therefore an extremely important technology. Thisrefolding has thus been under very active investigation, and, whiledifferent methods have been proposed, almost all of these methods have alow refolding rate and frequently can do nothing more than sporadicallygive desirable results for certain limited proteins (specific lowmolecular weight proteins in particular). At present there is no methodfor carrying out this refolding that is economical and efficient, thatprovides a high refolding rate, and that is a versatile and generalmethod applicable to variety of proteins.

Dialysis and dilution are the refolding techniques that have long beenin the most frequent use. In the former technique, the protein isdissolved in an aqueous solution that contains detergent and/ordenaturant, and this is dialyzed with buffer lacking detergent anddenaturant in order to reduce the detergent and/or denaturantconcentrations and refold the protein (typical example: the FoldIt Kitfrom Hampton Research Corporation). In the latter technique, the proteinis dissolved in an aqueous solution containing detergent and/ordenaturant and this is simply diluted in order to reduce the detergentand/or denaturant concentrations and induce refolding (typical example:the FoldIt Kit from Hampton Research Corporation). While these are thetechniques in general use, there are also other methods for inducingrefolding using a diluent, for example, a refolding method in which aglutathione S-transferase fusion protein is dissolved in a solution ofsodium N-lauroyl sarcosinate detergent and this is diluted with 1 to 2%Triton X-100 (refer to Anal. Biochem. Vol. 210 (1993) 179-187).

A consumable kit is commercially available from Hampton ResearchCorporation for both dialysis and dilution. Nothing more has been seenfor these protocols than the generation of refolding for a very limitednumber of proteins, such as ligand binding domains for glutamate andkainite receptors, lysozyme, and carbonic anhydrase B (refer to ProteinSci. Vol. 8 (1999): 1475-1483), and it is no exaggeration to say thatthey remain in the realm of trial and error methods. Thus, even when amethod occasionally goes smoothly, it is quite often the case that italmost never goes well when applied to another protein.

The use of an adsorption separation column for refolding has also beenattempted. Refolding is produced during gel filtration when thioredoxinprotein denatured by guanidine hydrochloride or urea is subjected to gelfiltration (refer to Biochemistry, Vol. 26 (1987) 3135-3141). However,refolding by this method is not always satisfactory, and satisfactoryresults are usually not obtained with other proteins. The eluted proteinis refolded when protein solubilized with 8 M urea is adsorbed on acolumn on which the molecular chaperone GroEL (a molecular chaperone isa type of protein that promotes the refolding of structure-disruptedprotein) has been immobilized and is eluted with a solution thatcontains 2 M potassium chloride and 2 M urea (refer to Proc. Natl. Acad.Sci. USA, Vol. 94 (1997) 3576-3578). However, this is confined to ademonstration for a very limited number of proteins, such as cyclophilinA. In particular, the use of molecular chaperones involves a certaintype of template, and this approach is in fact completely useless formaterial not conforming to the shape of this template.

Protein refolding on the resin has also been reported to occur whenguanidine hydrochloride-denatured scorpion toxin Cn5 protein is mixedwith resin on which three proteins thought to be related to refoldingpromotion (GroEL, disulfide oxidereductase from Escherichia coli (DsbA),and human proline cis-trans isomerase (PPI)) are simultaneouslyimmobilized (refer to Nat. Biotechnol. Vol. 17 (1999) 187-191). However,in addition to the drawback that this approach can be applied only tospecific proteins, such as scorpion toxin Cn5, the preparation of thethree protein-functionalized resin is complex and expensive.

Metal chelates have also been used in place of refolding protein as thematerial immobilized on a column. His6-tagged fusion protein undergoesrefolding when, after dissolution and denaturation with an aqueoussolution containing guanidine hydrochloride and urea and adsorption ontoa resin on which a nickel chelate has been immobilized, washing iscarried out with a buffer solution lacking denaturant (Life Science News(Japan Ed.) Vol. 3 (2001) 6-7). Again, the application of this method islimited to this protein and preparation of the resin is complex andexpensive.

Protein refolding has also been reported with the use ofbeta-cyclodextrin and cycloamylose as artificial chaperones. Whendetergent-denatured protein is mixed into a solution of such achaperone, the detergent is incorporated and sequestered by theartificial chaperone and the protein undergoes refolding during thisprocess (J. Am. Chem. Soc. Vol. 117 (1995) 2373-2374; J. Biol. Chem.Vol. 271 (1996) 3478-3487; and FEBS Lett. Vol. 486 (2000) 131-135). Thesuccess of this method, however, is confined to, for example, carbonicanhydrase B. Moreover, it is not a method that can be carried outrepetitively and it is therefore expensive.

The inventors, on the other hand, have continued to carry out researchup to the present time on the adsorption of biopolymers to zeolites(Chem. Eur. J., Vol. 7 (2001) 1555-1560), such as ZSM zeolite andzeolite beta (for example, refer to Zeolites, Vol. 11 (1991) 842-845;Adv. Mater., Vol. 8 (1996) 517-520; Japanese Laid-Open PatentApplication No. H06-127937; and Japanese Laid-Open Patent ApplicationNo. H08-319112).

DISCLOSURE OF THE INVENTION

While a variety of refolding methods have already been reported asdescribed above, the problems cited above are also associated with thesemethods, and a pressing issue in this area of technology has thereforebeen the development of a highly efficient, low-cost refolding methodthat enables repeated use, that is very versatile and generalizable, andthat can be applied, regardless of chain length, to a variety ofproteins that have either been denatured and deactivated or whose higherorder structure has yet to be formed. In addition, while a variety ofrefolding methods as well as substances and materials that have arefolding activity have already been reported and refolding kits madetherefrom have already been commercialized, the problems cited above arenevertheless still associated with these methods, substances, materials,and kits, and a pressing issue in this area of technology has thereforebeen the development of a highly efficient, low-cost refoldingsubstance, material, and method that enable repeated use, that are veryversatile and generalizable, and that can be applied, regardless ofchain length, to a variety of proteins that have either been denaturedand deactivated or whose higher order structure has yet to be formed, inother words, the development of an economical, high-performancerefolding technology and refolding kit based thereon. Moreover, while avariety of refolding methods as well as substances and materials thathave a refolding activity have been reported to date, these still sufferfrom the various problems described hereinabove, such as a lack ofgeneralizability, cumbersome procedures, and high cost. Due to this, apressing issue in this area of technology has therefore been, first ofall, the development of a highly efficient, low-cost refoldingsubstance, material, and method that enable repeated use, that are veryversatile and generalizable, and that can be applied, regardless ofchain length, to a variety of proteins that have either been denaturedand deactivated or whose higher order structure has yet to be formed. Asecond major issue has been that the prior refolding protocols andprocesses have required the use of a centrifugal separator andchromatography, and the repeated use thereof makes these protocols andprocesses complex and cumbersome, very time consuming, and expensive.

Against these circumstances and in view of the prior art as describedabove, the inventors carried out focused research and development forthe purpose of developing a novel refolding technology capable ofsolving the problems described hereinabove and also carried out detailedinvestigations into the nature of the adsorption of biopolymers, e.g.,DNA, RNA, protein, to metal oxides, such as zeolites (Chem. Eur. J. Vol.7 (2001) 1555-1560), as well as focused research on methods for theseparation and purification of protein. As a result of these campaigns,the inventors discovered that when protein produced, for example, by anEscherichia coli expression system, with an as yet unformed higher orderstructure, or protein deactivated for some reason, such as its thermalhistory, is treated with zeolite beta, such protein will exhibit itsnative function and activity. The inventors also discovered that thismethod can be used as a highly versatile, highly generalizable methodaccording to the present invention that is applicable to the refoldingof a variety of conformation-disordered proteins, including largeproteins with molecular weights in excess of 100,000. This invention wasachieved based on these discoveries. The inventors also discovered thatzeolite with the BEA structure, that is, zeolite beta, has a refoldingactivity for denatured protein, and concomitant with the development ofa refolding agent comprising zeolite beta, the inventors also developeda protein refolding kit in which this refolding agent is an essentialconstituent component. It was additionally found that this refoldingagent can also be applied to the refolding of a variety ofconformation-disordered proteins, including large proteins withmolecular weights in excess of 100,000, such as protein produced, forexample, by an Escherichia coli expression system, with an as yetunformed higher order structure, or protein deactivated for some reason,such as its thermal history, and this invention, that is, a highlyversatile and highly generalizable protein refolding technology andrefolding kit, was achieved thereby. An object of a first aspect of thisinvention is to provide a method for activating protein function. Anobject of a second aspect of this invention is to provide a novelrefolding kit. An object of a third aspect of this invention is toprovide a novel protein refolding material. An object of a fourth aspectof this invention is to provide a refolding molding comprising a moldingthat contains zeolite with the BEA structure and to provide a refoldingmolding that, by inducing the refolding of inactive protein, has thecapacity to activate or regenerate a native function inherent to theprotein.

A first aspect of the present invention is described in additionaldetail below.

The first aspect of the present invention comprises the followingtechnical means.

(1) A method of activating the function of a protein that is inactivedue to a disordered higher order structure, comprising bringing theprotein into a state that can express a native function inherent to theprotein, by bringing the protein into contact with zeolite beta.

(2) The method according to (1), wherein the protein is brought intocontact with the zeolite beta in the presence of a protein denaturant, asurfactant, and/or a refolding buffer.

(3) The method according to (1), wherein the protein that is inactivedue to a disordered higher order structure is a protein that is producedby an Escherichia coli expression system.

(4) The method according to (1), wherein the protein that is inactivedue to a disordered higher order structure is a protein that isdeactivated due to its thermal history.

(5) The method according to (1), wherein the protein is adsorbed to thezeolite beta by mixing with a solution that contains the zeolite beta orby introduction onto a column packed with the zeolite beta and is thendesorbed from the zeolite beta.

(6) A method for reforming the core structure of a protein, comprisingrefolding the conformation of a protein that is inactive due to adisordered higher order structure by bringing the protein into contactwith zeolite beta.

(7) A method for producing an active protein, comprising refolding theconformation of a protein that is inactive due to a disordered higherorder structure by bringing the protein into contact with zeolite beta,thereby producing a protein that has a controlled higher order structureand an activated native function inherent to the protein.

(8) The method according to (7) for producing a protein, comprisingrefolding the conformation of an inactive protein produced byEscherichia coli that incorporates the genetic code responsible for thesynthesis of a target protein, by bringing the inactive protein intocontact with zeolite beta.

Protein for submission to function activation according to the presentinvention generally comprises conformation-disordered protein producedby, for example, an Escherichia coli expression system and known as aninclusion body, as well as protein deactivated for some reason, such asthe thermal history. In accordance with the present invention, a nativefunction inherent to this protein is activated by refolding theconformation of the protein by treating the protein with zeolite beta.The activation protocol is typically carried out by first dispersing anddissolving the protein in a solution containing, for example, denaturantand/or detergent (surfactant); thereafter adsorbing the protein tozeolite beta by mixing with a zeolite beta-containing solution or byintroduction onto a column packed with zeolite beta; and then desorbingthe protein from the zeolite beta. The zeolite beta used as the functionactivator in the present invention can be exemplified by uncalcinedzeolite beta and by calcined zeolite beta obtained by the calcination ofsynthetic zeolite beta for 3 to 10 hours at 300 to 500° C. However, theinvention is not limited to these and zeolite beta equivalent to thepreceding can be similarly used.

Given that in general the protein is frequently produced in, forexample, an Escherichia coli expression system, and is typicallyfrequently used in aqueous solution, and that the deactivated proteinfrequently resides in aqueous solution, water, for example, is verysuitably used as the solvent for dispersing the protein prior toadsorption to the zeolite beta. However, the solvent is not necessarilylimited to this, and solvents can be used, either as such or mixed withwater, that do not react with the protein or cause the conformation ofthe protein to change to an unintended shape and thus that are basicallyfree of problems. Typical examples of solvents of this type aremonovalent and polyvalent alcohols, but there is no restriction tothese.

The subject protein adsorption/desorption is generally carried out inthe presence of denaturant and/or detergent, pH regulator, refoldingfactor, and so forth in order to facilitate unraveling of the entangledprotein chain, e.g., an inclusion body, and facilitate its refolding,and/or in the presence of some type of reducing agent in order to cleaveS—S bonds unintentionally formed in the protein chain. Typical examplesof these denaturants and/or detergents, pH regulators, and refoldingfactors are guanidine hydrochloride, trisaminomethane hydrochloride,polyethylene glycol, cyclodextrin,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),polyphosphoric acid, sucrose, glucose, glycerol, inositol, DextranT-500, and Ficol1400, but these substances are not confined to thepreceding and any substance that has an equivalent action can be used.

2-mercaptoethanol, because it is inexpensive and easy to acquire, istypically used as the reducing agent that supports return to the nativestructure by cleavage of unintentionally formed S—S bonds; however, thisreducing agent is not limited thereto and any substance that has anequivalent activity can be used. Of course, when unraveling of theprotein chain proceeds easily or when there is no unintended S—S bondproduction, use of the denaturant and/or detergent and/or inhibitor isnot necessarily required and their presence therefore is not alwaysrequired; rather, their use should be selected as appropriate incorrespondence to the circumstances. When these substances are used,their quantities are determined as appropriate to the circumstances.

Displacement adsorption is generally used for desorption of the protein;however, there are no particular limitations here as basically anyprocedure can be used that does not impair activation of the functionafter desorption of the protein. Thus, changes in the pH or temperaturecan also be used, and these can also be used in combination withdisplacement adsorption. A detergent such as sodium dodecyl sulfate(SDS) or a salt such as an alkali halide is generally used as thesubstance that induces desorption of the protein by displacementadsorption, but there is no limitation to these. Insofar as there is noimpairment of activation of the function after desorption of theprotein, a variety of substances can generally be used, such as thesubstances used for elution in column chromatography.

Various supplementary procedures can also be carried out in combinationwith the aforementioned protocol in order to induce adsorption of theprotein to the silicate or desorption therefrom. A typical example ofsuch a procedure involves, for example, exposure to ultrasound ormicrowaves and/or application of a magnetic or electrical field. Theprocedures and protocol according to the present invention as describedabove cause the refolding of protein produced using, for example, anEscherichia coli expression system, that has an as yet unformed higherorder structure, and cause the refolding of protein deactivated for somereason, and thereby rapidly activate a native function of such proteins.The zeolite beta function activator according to the present inventionis very stable both thermally and chemically and is inexpensive and inaddition can be used repeatedly. This invention is extremely useful forthe production of biochemical and pharmaceutical products and hasimmeasurable economic effects.

A second aspect of the present invention is described in additionaldetail below.

The second aspect of the present invention comprises the followingtechnical means.

(1) A protein refolding kit that is a reagent kit used in a proteinfunction activation (refolding) protocol or step that modulates thehigher order structure of a protein that is inactive due to a disorderedhigher order structure, thereby activating the protein, wherein theprotein refolding kit contains a refolding agent comprising zeolite withthe BEA structure (zeolite beta) as a constituent.

(2) The refolding kit according to (1), wherein the kit has proteindenaturant, pH regulator, and refolding agent comprising theaforementioned zeolite beta as basic constituent components andadditionally comprises a combination that contains at least oneselection from agents that inhibit the formation of protein S—S bridges,surfactants, and refolding factors.

(3) The refolding kit according to (1) or (2), wherein the frameworkstructure of the zeolite beta contains silicon, oxygen, and at least oneelement other than silicon and oxygen.

(4) The refolding kit according to any of (1) to (3), wherein theframework structure of the zeolite beta comprises only silicon andoxygen or only silicon and aluminum and oxygen.

(5) The refolding kit according to any of (1) to (4), wherein thezeolite beta contains an ammonium species.

(6) The refolding kit according to (5), wherein the ammonium species isammonium ion, an organic amine, and/or an acid amide.

(7) The refolding kit according to (6), wherein the organic amine is atetraalkylammonium.

(8) The refolding kit according to any of (2) to (7), wherein theprotein denaturant in the kit is guanidine hydrochloride.

(9) The refolding kit according to any of (2) to (8), wherein the pHregulator in the kit is trisaminomethane trihydrochloride (TrisHCl)and/or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

(10) The refolding kit according to any of (2) to (9), wherein the agentin the kit that inhibits the formation of protein S—S bridges is2-mercaptoethanol, dithiothreitol, cystine, or thiophenol.

(11) The refolding kit according to any of (2) to (10), wherein thesurfactant and refolding factor in the kit are at least one selectionfrom polyethylene glycol, Ficol170, Ficol1400, polyphosphoric acid,sodium dodecyl sulfate (SDS), sucrose, glucose, glycerol, inositol,cyclodextrin, amylose, Dextran T-500, Tween 20, Tween 40, Tween 60,NP-40, SB3-14, SB12, CTAB, and Triton X-100.

(12) The refolding kit according to any of (1) to (7), wherein the kitcomprises the aforementioned refolding agent, guanidine hydrochloride,TrisHCl, 2-mercaptoethanol, and a solution (the refolding buffer)comprising HEPES, alkali halide, 2-mercaptoethanol, refolding factor,and surfactant, or the kit comprises the refolding buffer, the refoldingagent, guanidine hydrochloride, TrisHCl, 2-mercapethanol, and alkalihalide.

Protein that may be processed by the reagent set, that is, the refoldingkit, according to the present invention for activation of a function ofan inactive protein is any inactive protein with an irregular higherorder structure, but generally will be conformation-disordered proteinproduced by, for example, an Escherichia coli expression system andknown as an inclusion body, or protein deactivated for some reason, suchas the thermal history. The kit according to the present inventioneffects activation or generation of a native function of a protein byrefolding the conformation of the protein by a process in which theprotein is adsorbed to and desorbed from a refolding agent comprisingzeolite beta that is present in the kit. However, the subject capabilityof the refolding agent is not necessarily limited to the preceding andis generally manifested by the following protocol. In other words,activation of a function of an inactive protein is carried out by thefollowing protocol. That is, this protocol is carried out by a sequencein which the protein is first dissolved and dispersed in a solutioncontaining denaturant and/or detergent (surfactant) and so forth; thisis mixed with a solution containing the refolding agent, or isintroduced onto a column packed with the refolding agent, in order toadsorb the protein to the refolding agent; and the protein is thendesorbed from the refolding agent.

In addition to the refolding agent comprising zeolite beta, the kitaccording to the present invention suitably contains, for example,denaturant and/or pH regulator and, in addition to the preceding,comprises refolding factor and/or detergent and S—S bridge formationinhibitor in order to prevent reformation of the inclusion body afterrefolding and promote desorption of the protein from the refoldingagent.

A typical example of this type of denaturant in the kit according to thepresent invention is guanidine hydrochloride, while typical examples ofthe pH regulator in the kit according to the present invention aretrisaminomethane hydrochloride (TrisHCl) and4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). However, thekit according to the present invention is not limited to these, and anysubstance with the equivalent activity can be used.

Typical examples of the refolding factor and detergent in the kitaccording to the present invention are polyethylene glycol (PEG20K,PEG800, PEG200, PEG3350), Ficol170, Ficol1400, polyphosphoric acid,sodium dodecyl sulfate (SDS), sucrose, glucose, glycerol, inositol,cyclodextrin, amylose, Dextran T-500, Tween 20, Tween 40, Tween 60,NP-40, SB3-14, SB12, CTAB, and Triton X-100. However, the kit accordingto the present invention is not limited to these, and any substance withthe equivalent activity can be used.

Of course, when unraveling of the protein chain proceeds easily or whenthere is no unintended S—S bridge production, use of the denaturantand/or detergent and/or inhibitor is not necessarily required and theirpresence therefore is not always required; rather, their use in the kitshould be selected as appropriate in correspondence to the circumstancesof use. In addition, the amounts of the particular components making upthe kit are determined as appropriate for the circumstances. The kitaccording to the present invention preferably generally containsrefolding agent comprising zeolite beta, guanidine hydrochloride(denaturant), and TrisHCl and HEPES (pH regulators) and also one or moreselections from 2-mercaptoethanol and the aforementioned refoldingfactors and detergents.

In general, displacement adsorption by a reagent (refolding factorand/or detergent) present in the subject kit is ordinarily used forprotein desorption during refolding procedures and operations using thekit. However, basically any procedure that does not impair activation ofthe function after protein desorption is usable here and there areotherwise no particular restrictions thereon. Thus, changes in the pH ortemperature can be used, and these can also be used in combination withdisplacement adsorption.

The salts, e.g., alkali halides, heretofore used for elution in columnchromatography are frequently used as a substance that inducesdesorption of the protein during displacement adsorption, and remarkableresults are frequently also obtained by the co-use thereof. Moreover,with regard to actual use of the reagents making up the kit according tothe present invention, some of the kit reagents can also be combined inadvance to make a single reagent, insofar as there are no particularproblems or obstacles, such as the occurrence of a reaction between oramong the kit reagents. For example, a single solution comprising HEPES,alkali halide (for example, sodium chloride), 2-mercaptoethanol,refolding factor (for example, beta-cyclodextrin or Ficol170 and/or aselection from the polyethylene glycols PEG20K, PEG800, PEG200, andPEG3350), and detergent (for example, Tween 20, Tween 40, Tween 60,NP-40, or Triton X-100) can be made up as a refolding buffer and thiscan also be provided as one of the reagents making up the kit accordingto the present invention. This is quite often very convenient for therefolding protocol and steps.

Various supplementary procedures can also be carried out in combinationwith the aforementioned protocol in order to induce adsorption of theprotein to the refolding agent in the kit or desorption therefrom. Atypical example of such a procedure involves, for example, exposure toultrasound or microwaves and/or application of a magnetic or electricalfield. The aforementioned procedures and protocol using the kitaccording to the present invention cause the protein refolding capacityof the subject refolding agent to be strongly expressed, resulting inthe refolding of protein produced using, for example, an Escherichiacoli expression system, that has an as yet unformed higher orderstructure, or the refolding of protein deactivated for some reason, andthereby rapidly activating functionality that should be native to suchproteins.

Typical examples of the zeolite with the BEA structure, that is, zeolitebeta, that constitutes the refolding agent which expresses an activationfunction for inactive protein and a refolding capacity for denaturedprotein are the usual commercially available zeolite betas (for example,HSZ-930NHA from Tosoh Corporation); zeolite beta as synthesized orproduced inhouse in accordance with directions found in the literature(refer to Zeolites, Vol. 11 (1991) 202); zeolite beta obtained bycalcination of the preceding examples; zeolite beta in which an ammoniumspecies (e.g., ammonium, various aliphatic and/or aromatic ammoniumspecies) resides in the zeolite cavities; framework-substituted zeolitebeta in which a portion of the framework silicon making up the zeolitehas been substituted by another metal; and framework-substituted zeolitebeta containing the aforementioned ammonium. As long as the frameworkstructure of zeolite beta is present, zeolite beta having all of theaforementioned function and activity that makes up the subject refoldingagent is basically not necessarily restricted to those provided asexamples hereinabove.

The aforementioned function and activity of the subject refolding agentare expressed by bringing inactive or denatured protein into contactwith the refolding agent, that is, by adsorption and desorption. Duringthis process, the affinity between the surface of the refolding agentand the target protein is important, and in addition to this it isfrequently also the case that protein adsorption/desorption isinfluenced by the dispersing solvent therefor, the denaturant,surfactant, and refolding factor in the dispersing solvent, the pH ofthe dispersing solvent, and so forth. As a consequence, the refoldingactivity of the refolding agent with respect to the target protein andthe composition of the solution containing the target protein frequentlyvaries among the different zeolite betas described above that can makeup the refolding agent. As a rule, however, refolding agent comprisingzeolite beta that contains an ammonium species has a higher refoldingactivity than in the absence of the ammonium species, and for thisreason the use of refolding agent comprising zeolite beta containing anammonium species and the use of refolding agent comprisingframework-substituted zeolite beta containing an ammonium species isfrequently preferred.

The ammonium species that should be present in the zeolite beta thatmakes up the subject refolding agent can be an ammonium species thattends to remain in the cavities present in the zeolite, for example, theammonium ion; mono-, di-, tri-, and tetraalkylammonium ions where thealkyl is methyl, ethyl, propyl, butyl, and so forth; the ammonium ionsof 5-, 6-, and 7-member cyclic aliphatic and aromatic amines and moreparticularly the piperidium ion, alkylpiperidium ion, pyridinium ion,alkylpyridinium ion, aniline ion, and N-alkylaniline ion; and formamide,acetamide, and their N-alkyl substitution products as examples of acidamides. However, basically any ammonium species that can enter the porespresent in the zeolite beta can be used and there is no restriction tothe ammonium species provided as examples hereabove.

The elements forming the framework of the zeolite beta that makes up thesubject refolding agent are generally silicon and oxygen or silicon,oxygen, and aluminum; however, zeolite beta in which a portion of thesilicon or aluminum has been substituted by another element andsubstituted zeolite beta containing the aforementioned ammonium speciesin the pores thereof can also provide refolding agent that effectsfunction activation on an inactive protein. Typical examples of elementsthat can substitute for the framework silicon in zeolite beta arealuminum, boron, phosphorus, gallium, tin, titanium, iron, cobalt,copper, nickel, zinc, chromium, and vanadium, but there is no limitationto the preceding and basically any element that does not destroy thezeolite beta structure can be used. With regard to the amount ofsubstitution, any amount of substitution that does not destroy thezeolite beta structure is unproblematic and the subject substitutedzeolite beta will have the same ability to provide a refolding agent forinactive or denatured protein. With regard to other features of thepresent invention, the items described for the first aspect of thepresent invention are also similarly applied to the present invention.

A third aspect of the present invention is described in additionaldetail below.

The third aspect of the present invention comprises the followingtechnical means.

(1) A protein refolding agent that has a protein refolding action thatmodulates the higher order structure of and activates a protein that isinactive due to a disordered higher order structure, the proteinrefolding agent comprising zeolite with the BEA structure (zeolitebeta).

(2) The refolding agent according to (1), that carries out proteinrefolding in the presence of a protein denaturant, a surfactant, and/ora refolding buffer.

(3) The refolding agent according to (1), wherein the protein that isinactive due to a disordered higher order structure is a protein that isproduced by an Escherichia coli expression system.

(4) The refolding agent according to (1), wherein the protein that isinactive due to a disordered higher order structure is a protein that isdeactivated due to its thermal history.

(5) The refolding agent according to (1), wherein the zeolite betacontains ammonium ion, an organic ammonium ion, and/or urea.

(6) The refolding agent according to (5), wherein the organic ammoniumion is a mono-, di-, tri-, and/or tetraalkylammonium ion (where thealkyl group is methyl, ethyl, propyl, or butyl).

(7) The refolding agent according to (1), wherein the frameworkstructure of the zeolite beta comprises oxygen and at least one elementother than oxygen.

(8) The refolding agent according to (7), wherein the frameworkstructure of the zeolite beta comprises silicon and oxygen or silicon,aluminum, and oxygen.

(9) The refolding agent according to any of (1) to (8), that manifests aprotein refolding action through contact with a protein dispersed in asolution.

(10) The refolding agent according to any of (1) to (9), that causesrefolding of the protein by a procedure in which the protein in asolution is adsorbed by mixing with the refolding agent or byintroduction onto a column packed with the refolding agent andthereafter is desorbed.

Protein that may be processed by the refolding agent according to thepresent invention comprising zeolite beta is any inactive protein withan irregular higher order structure, but in particular will beconformation-disordered protein produced by, for example, an Escherichiacoli expression system and known as an inclusion body, or proteindeactivated for some reason, such as the thermal history. The refoldingagent according to the present invention effects activation orgeneration of a native function of a protein by refolding theconformation of the protein by a process in which the protein isadsorbed to and desorbed from the refolding agent. However, the subjectcapability of the refolding agent is not necessarily limited to thepreceding and is generally manifested by the following protocol. Inother words, activation of a function of an inactive protein is carriedout by the following protocol. That is, this protocol is carried out bya sequence in which the protein is first dissolved and dispersed in asolution containing denaturant and/or detergent and so forth; this ismixed with a solution containing the refolding agent according to thepresent invention, or is introduced onto a column packed with therefolding agent, in order to adsorb the protein to the refolding agent;and the protein is then desorbed from the refolding agent.

Displacement adsorption is generally used for desorption of the protein;however, there are no particular limitations here as basically anyprocedure can be used that does not impair activation of the functionafter desorption of the protein. Thus, changes in the pH or temperaturecan also be used, and these can also be used in combination withdisplacement adsorption. A salt as heretofore used for elution in columnchromatography, such as sodium dodecyl sulfate or an alkali halide, isoften used as the substance that induces desorption of the proteinduring displacement adsorption, and their co-use frequently alsoprovides remarkable results. Accordingly, it is also possible duringexecution of protein desorption by displacement desorption to use thecombination of various salts, such as those used for elution in columnchromatography, with surfactant and/or refolding factor. These saltsused in combination are not limited to those provided as examples here,and any salt can be used as long as it does not impair the activation offunction after desorption of the protein.

Various supplementary procedures can also be carried out in combinationwith the aforementioned protocol in order to induce adsorption of theprotein to the refolding agent according to the present invention ordesorption therefrom. A typical example of such a procedure involves,for example, exposure to ultrasound or microwaves and/or application ofa magnetic or electrical field. The protein refolding activity of therefolding agent is strongly manifested through the procedures andprotocol as described above, causing the refolding of protein producedusing, e.g., an Escherichia coli expression system, that has an as yetunformed higher order structure, or causing the refolding of proteindeactivated for some reason, and thereby rapidly activating a nativefunction that the protein should have. With regard to other features ofthe present invention, the items described for the first aspect of thepresent invention are also similarly applied to the present invention.

A fourth aspect of the present invention is described in additionaldetail below.

The fourth aspect of the present invention comprises the followingtechnical means.

(1) A refolding molding comprising a molding that contains zeolite withthe BEA structure (known as zeolite beta) that has the capacity, denotedas a refolding activity, to modulate and activate the higher orderstructure of a protein that is inactive due to a disordered higher orderstructure.

(2) The refolding molding according to (1), wherein the moldingcomprises zeolite beta or zeolite beta and a substrate that supports thezeolite beta.

(3) The refolding molding according to (1), that manifests a refoldingactivity upon contact with a protein.

(4) The refolding molding according to (1), that carries out therefolding of a protein in the presence of a protein denaturant, asurfactant, and/or a refolding buffer.

(5) The refolding molding according to (1), wherein the protein that isinactive due to a disordered higher order structure is a protein that isproduced by an Escherichia coli expression system.

(6) The refolding molding according to (1), wherein the protein that isinactive due to a disordered higher order structure is a proteindeactivated due to its thermal history.

(7) The refolding molding according to (1), wherein the zeolite betacontains ammonium ion and/or organic ammonium ion.

(8) The refolding molding according to (7), wherein the organic ammoniumis a mono-, di-, tri-, and/or tetraalkylammonium ion (where the alkylgroup is methyl, ethyl, propyl, or butyl).

(9) The refolding molding according to (1), wherein the frameworkstructure of the zeolite beta comprises oxygen and at least one elementother than oxygen.

(10) The refolding molding according to (9), wherein the frameworkstructure of the zeolite beta comprises silicon and oxygen or silicon,aluminum, and oxygen.

(11) The refolding molding according to any of (1) to (10), thatmanifests a protein refolding activity through contact with a proteindispersed in a solution.

(12) The refolding molding according to any of (1) to (11), that has afunction that causes refolding of the protein by a procedure in whichthe protein is adsorbed to the molding by mixing the protein in asolution with the refolding molding or by flowing or dripping theprotein in a solution onto the molding and thereafter is desorbed.

The refolding molding according to the present invention comprises justzeolite with the BEA structure, so-called zeolite beta, or compriseszeolite beta and a substrate (support) that supports the zeolite beta. Asupport is absent in the former case, while in the latter case a supportis attached. As a general matter, the substances known as zeolites areoften difficult to mold by themselves due to their poorself-sinterability. As a consequence, insofar as concerns thefabrication of such moldings, that is, the design and control of theirshape and configuration, the latter case, because it enablesimmobilization and/or coating of the zeolite beta on a support with apre-arranged shape, in general frequently provides greater latitude andis more advantageous than the former case.

However, the method of fabrication, which begins with the decision onwhether to use a support for the molding and includes the design andcontrol of the shape and configuration, varies as a function of how themolding will be utilized and the configuration of use and thus isselected as appropriate. Accordingly, the known methods are all usablefor fabrication of the molding under consideration and may be selectedas appropriate, and combinations of these methods can also be used, andas a consequence there are no particular restrictions on fabrication ofthis molding and in particular a detailed description or discussion isunnecessary. This notwithstanding, several examples will be providedhereinbelow of methods for fabricating the molding under consideration,that is, methods for designing and controlling the shape andconfiguration. Typical conventional methods for the fabrication ofzeolite moldings, i.e., in situ zeolite synthesis, dry gel conversion,solid-phase conversion (refer to Stud. Surf. Sci. Catal. Vol. 125 (1999)1-12; Hyomen [Surface], Vol. 37 (1999) 537-557), can also be used forfabrication of the molding under consideration, both for thesupport-free version and the support-attached version. Methods usablefor the fabrication of support-attached moldings include incorporationinto an organic polymer (Zeolites, Vol. 16 (1996) 70), bonding/moldingof the zeolite beta by means of an inorganic powder such as alumina, andimmobilization of the zeolite beta on the support by means of awater-insoluble adhesive, but there is no limitation to the preceding.

The shape and configuration of the molding under consideration isselected as appropriate from, for example, chip shapes, film or membraneshapes, pellet shapes, and bead shapes, in correspondence to how themolding will be utilized and the configuration of use. In the particularcase of the support-attached moldings under consideration, the zeolitebeta can be immobilized and/or coated by the aforementioned methods onsupports with a variety of shapes, for example, plates, spheres,cylinders, tubes, columns, troughs, and U-shaped channels. This offersthe advantage of enabling the molding to be executed in any desiredshape. The support in this case can be exemplified by glass; quartz;various ceramics such as alumina, silica, cordierite, and mullite;cellulosics such as paper; and various organic polymers such as Teflon®,nylon, polyethylene, polypropylene, and polyethylene terephthalate(PET). However, basically any support is acceptable that is waterinsoluble and that does not negatively affect protein and the support isnot limited to those provided above as examples.

Protein that may be processed by the refolding molding according to thepresent invention is any inactive protein with an irregular higher orderstructure, but in particular will be conformation-disordered proteinproduced by, for example, an Escherichia coli expression system andknown as an inclusion body, or protein deactivated for some reason, suchas the thermal history. The refolding molding according to the presentinvention effects activation or generation of a native function of aprotein by refolding the conformation of the protein by a process inwhich the protein is adsorbed to and desorbed from the refoldingmolding. However, the subject capability of the refolding molding is notnecessarily limited to the preceding and is generally manifested by thefollowing protocol. In other words, activation of a function of aninactive protein is carried out by the following protocol. This protocolis carried out by a sequence in which the protein is first dissolved anddispersed in a solution containing denaturant and/or detergent and soforth; this solution is then mixed with the refolding molding accordingto the present invention or is poured onto, flowed into or over, ordripped onto the refolding molding, in order to adsorb the protein tothe refolding molding; and the protein is then desorbed from therefolding molding. There is no specific requirement for a separator,such as a centrifugal separator, in these steps. With regard to otherfeatures of the present invention, the items described for the firstaspect of the present invention are also similarly applied to thepresent invention.

The present invention relates, inter alia, to a method for activating afunction of an inactive protein, and the following effects are achievedby the present invention:

1) a native function or activity of a protein produced by, for example,an Escherichia coli expression system and being inactive due to an asyet unformed higher order structure, or of a protein deactivated due toa change in its conformation for some reason, can be activated byrefolding;

2) this method is useful for the highly efficient refolding of inclusionbodies;

3) an efficient method is provided that has a high refolding rate, thatis versatile and generalizable, and that is applicable to a variety ofproteins;

4) the zeolite beta comprising the function activator used by thepresent invention is inexpensive and can be used repeatedly;

5) this method can be applied to the refolding of a variety ofconformation-disordered proteins, including large proteins withmolecular weights in excess of 100,000; and

6) the combination of the method according to the present inventionwith, for example, a protein synthesis process based on an Escherichiacoli expression system, enables the elaboration of a novel process formanufacturing active protein that produces protein having a controlledhigher order structure and a native function inherent to the protein.

The invention additionally relates to a reagent kit that is used in theprocedures and/or processes for activation of a function of an inactiveprotein and further relates to a method of using this reagent kit. Thefollowing separate effects are achieved by the present invention:

1) an all-purpose reagent set, that is, an all-purpose kit, can beselected that can activate a native function of a wide range of inactiveproteins, regardless of the type of protein;

2) the use of this kit to treat such protein, for example, a proteinproduced by, for example, an Escherichia coli expression system andbeing inactive due to an as yet unformed higher order structure, or aprotein deactivated due to a change in its conformation for some reason,enables the activation of a native function or activity of the proteinby refolding;

3) the subject kit is also effective on the protein in inclusion bodiesand is useful in providing an efficient method for the refolding ofinclusion bodies utilizing this activity;

4) the method for activating the function of inactive protein throughthe use of this kit provides a versatile, generalizable, and efficientmethod that has a high refolding rate and that can be applied to avariety of denatured proteins regardless of the chain length andsequence of the amino acids making up the protein;

5) the zeolite with the BEA structure, that is, zeolite beta, that makesup the refolding agent that is an essential component of this kit isinexpensive and can be used repeatedly;

6) the method for activating the function of inactive protein utilizingthe subject kit can be applied to the refolding of a variety ofconformation-disordered proteins, including large proteins withmolecular weights in excess of 100,000; and

7) the combination of the activation of a function of an inactiveprotein using the subject kit with, for example, a protein synthesisprocess based on an Escherichia coli expression system, enables theelaboration and establishment of a novel process for manufacturingactive protein that produces protein that has a controlled higher orderstructure and that is provided with a native function inherent to theprotein.

The invention additionally relates to a refolding agent and a moldingthat are a substance and material that have the capacity to activate afunction of an inactive protein. The following separate effects areachieved by the present invention:

1) an all-purpose substance or material, that is, refolding agentcomprising zeolite with the BEA structure (zeolite beta), can beselected that can activate a native function of a wide range of inactiveproteins, regardless of the type of protein;

2) effecting contact between the refolding agent according to thepresent invention and such protein, for example, a protein produced by,for example, an Escherichia coli expression system and being inactivedue to an as yet unformed higher order structure, or a proteindeactivated due to a change in its conformation for some reason, enablesthe activation of a native function or activity of the protein byrefolding;

3) the refolding agent according to the present invention is alsoeffective on the protein in inclusion bodies and is useful in providingan efficient method for the refolding of inclusion bodies;

4) the method for activating the function of inactive protein throughcontact with the refolding agent according to the present invention is aversatile, generalizable, and efficient method that has a high refoldingrate and that can be applied to a variety of denatured proteinsregardless of the chain length and sequence of the amino acids making upthe protein;

5) the zeolite beta that makes up the refolding agent according to thepresent invention is inexpensive and can be used repeatedly;

6) the method for activating the function of inactive protein utilizingthe refolding agent according to the present invention can be applied tothe refolding of a variety of conformation-disordered proteins,including large proteins with molecular weights in excess of 100,000;and

7) the combination of the activation of a function of an inactiveprotein by the refolding agent according to the present invention with,for example, a protein synthesis process based on an Escherichia coliexpression system, enables the elaboration and establishment of a novelprocess for manufacturing active protein that produces protein that hasa controlled higher order structure and that is provided with a nativefunction inherent to the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an electrophoretic gel shift assay.

FIG. 2 shows the recovery rate and activity recovery rate for refoldedprotein.

FIG. 3 shows the results of an electrophoretic gel shift assay.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is specifically described hereinbelow based on examplesand comparative examples, but the invention is in no way limited by theexamples and other material that follow.

Examples and Comparative Examples for the First Aspect of the PresentInvention

The examples provided below describe the activation of a function ofprotein produced by an Escherichia coli expression system and denaturedprotein; however, the present invention is neither limited to norrestricted by these examples.

1) Preparation of Materials (a) The Function Activator

The following were used as the function activator: uncalcined zeolitebeta (Na-BEA) as shown in Table 1 below, synthetic zeolite beta as shownin Table 1, calcined zeolite beta obtained by calcination of thesynthetic zeolite, and comparative products as shown in Table 1 forComparative Examples 1 to 15.

(b) The Denatured Protein Solution

The proteins used are described under “protein” and “remarks” in Table 1and included RPA70 (Drosophila melanogaster origin) and P53 (humanorigin).

(c) The Refolding Buffer

The salt concentration of the refolding buffer was investigated usingNa-BEA as the zeolite and RPA70 as the protein. The refolding bufferused 20 mM TrisHCl pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol, 2.5%(w/v) polyethylene glycol 20,000, and nonionic detergent wherein 1%(v/v) Tween 20, Triton X-100, and NP-40 were used as the detergent. Thedetails of the refolding buffers actually used in the examples andcomparative examples are shown in Table 2 below.

2) The Refolding Protocol

100 mg function activator was introduced into a 1.5 mL Eppendorf tubefollowed by the addition of 0.5 mL 6 M guanidine hydrochloride, 20 mMtrisaminomethane trihydrochloride (TrisHCl) pH 7.5, 0.5 M NaCl, and 20mM 2-mercaptoethanol and suspension. To this was then added 6 Mguanidine hydrochloride and 20 mM 2-mercaptoethanol followed by holdingfor one hour on ice, after which 0.5 mL of the denatured proteinsolution (concentration from 0.5 to 1.0 mg/mL) was added. In order toensure adsorption of the protein on the function activator, this mixturewas stirred for 1 hour with a Rotary Culture RCC-100 (Iwaki Glass Co.,Ltd.) placed in a cold room.

The function activator was then sedimented by centrifugation for 5seconds at 10000×g and the supernatant was removed. In order tocompletely remove the protein denaturant from the sedimented functionactivator, it was washed 4 times with 1 mL 20 mM TrisHCl pH 7.5, 20 mM2-mercaptoethanol followed by centrifugation for 5 seconds at 10000×gand discard of the supernatant thereby produced. The remaining functionactivator was suspended by the addition thereto of 1 mL refolding buffer(comprising 50 mM HEPES pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol,refolding factor, and nonionic detergent).

In order to desorb and elute the protein adsorbed on the functionactivator, this suspension was again stirred in the cold with the RotaryCulture RCC-100 (Iwaki Glass Co., Ltd.). The function activator wasthereafter sedimented by centrifugation for 5 seconds at 10000×g, andthe protein-containing supernatant was transferred to a new Eppendorftube and this was used for activity measurement (assay).

Methods appropriate to the action of the protein used were employed forthe activity measurements. Specifically, the activity was measured usingthree types of measurements, i.e., a gel shift assay, a polymeraseassay, and measurement of lysozyme activity.

3) Activity Measurement Protocols (a) The Gel Shift Assay

1 pmol radioisotope-labeled DNA oligonucleotide and the refolded proteinwere incubated for 30 minutes on ice in a solution with a composition of25 mM HEPES pH 7.4, 50 mM KCl, 20% glycerol, 0.1% NP-40, 1 mM DTT, and 1mg/mL bovine serum albumin. This was followed by electrophoresis at 4°C. on 4.5% polyacrylamino gel using 0.5×TBE buffer. The results areshown in FIGS. 1 and 3.

When DNA binding to the protein was present (that is, when activity waspresent), the protein underwent binding to the DNA, which slowed downelectrophoresis and caused band shifting, thereby enabling adetermination of activity (that is, the refolding rate).

(b) The Polymerase Assay

Poly(dA)oligo(dT)₁₂₋₁₈ or DNase I-activated calf thymus DNA was used asthe template DNA. The reaction solution had a composition (finalconcentration) of 50 mM TrisHCl pH 7.5, 1 mM DTT, 15% glycerol, 5 mMMgCl₂, 0.5 μM dTTP (cold) (thymidine triphosphate), and [³H]-dTTP (5mCi/mL:100-500 cpm/pmol). The protein (enzyme) sample solution was firstadded to and suspended in 10 μL reaction solution that was twice asconcentrated as that given above followed by incubation for 1 hour at37° C., after which the reaction was stopped by holding on ice.

The reaction solution was then dripped onto DE81 paper that had been cutinto a square. After drying, this was transferred to a beaker and waswashed in order to dissolve and remove the unreacted dTTP. This washconsisted of first 3× with 5% aqueous disodium hydrogen phosphatesolution, then 3× with distilled water, then 2× with ethanol, and wasfollowed by drying. The dry DE81 paper obtained in this manner wasplaced in a scintillator-containing vial and the radioactivity (cpm) wasmeasured with a scintillation counter. Stronger activity by the enzymesample resulted in greater incorporation of radioisotope-labeled dTTP inthe thereby synthesized DNA and thus in greater radioactivity, and theprotein activity was determined on this basis. The refolded proteinrecovery rate and the activity recovery rate are shown in FIG. 2 for theuse of Tween 20.

(c) Measurement of Lysozyme Activity

The bacteria M. lysodeikticus was selected as the substrate and wassuspended in 50 mM phosphate buffer to prepare a substrate solution witha concentration of 0.16 mg/mL. 20 μL of the protein (lysozyme enzyme)solution was added to 480 μL of this substrate solution followed byincubation for 30 minutes at room temperature. This was followed bymeasurement of the absorbance at a wavelength of 450 nm.

Lysozyme has the capacity to degrade the cell wall of bacteria, and as aresult the higher this capacity, that is, the activity, the greater thedecline in absorbance. 1 unit of lysozyme activity was defined as adecline in absorbance at 450 nm of 0.001 per minute.

The activity (refolding rate) and protein recovery rate, which are theresults for the subject examples obtained by the procedures andprotocols described above, are shown in Table 1 in combination with theresults for the comparative examples. The refolding buffers used in theexamples and comparative examples are shown in Table 2. As shown by theexamples, activity native to the proteins, for example, DNA bindingactivity, is produced by refolding. The present invention is useful as ahighly versatile, highly generalizable refolding method that isapplicable to a variety of denatured and deactivated proteins andproteins that have an as yet unformed higher order structure; however,the application of the present invention is not limited to the proteinsshown in the examples and the present invention can be applied to anyprotein.

TABLE 1 function activator protein activity (refolding rate); proteinrecovery rate remarks Example 1 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (high) the RPA7Cused was synthesized and (commercial Na-BEA) origin) precipitated in E.coli, MW 66 kDa Example 2 uncalcined zeolite beta RPA70 (Drosophilamelanogaster DNA binding activity present (medium) (commercial Na-BEA)origin) Example 3 uncalcined zeolite beta RPA70 (Drosophila melanogasterDNA binding activity present (low) (commercial Na-BEA) origin) Example 4uncalcined zeolite beta RPA70 (Drosophila melanogaster DNA bindingactivity present (high): ca. 90%; 20% see FIG. 1 (commercial Na-BEA)origin) Example 5 uncalcined zeolite beta RPA70 (Drosophila melanogasterDNA binding activity present (high) see FIG. 1 (commercial Na-BEA)origin) Example 6 uncalcined zeolite beta RPA70 (Drosophila melanogasterDNA binding activity present (high) see FIG. 1 (commercial Na-BEA)origin) Example 7 uncalcined zeolite beta RPA70 (Drosophila melanogasterDNA binding activity present (high): ca. 80%; 16% see FIG. 2 (commercialNa-BEA) origin) Example 8 uncalcined zeolite beta RPA70 (Drosophilamelanogaster DNA binding activity present (high): a little over 95%; 23%see FIG. 2 (commercial Na-BEA) origin) Example 9 uncalcined zeolite betaRPA70 (Drosophila melanogaster DNA binding activity present (high): ca.95%; 22% see FIG. 2 (commercial Na-BEA) origin) Example 10 uncalcinedzeolite beta RPA70 (Drosophila melanogaster DNA binding activity present(high): a little over 90%; 19% see FIG. 2 (commercial Na-BEA) origin)Example 11 uncalcined zeolite beta RPA70 (Drosophila melanogaster DNAbinding activity present (low) see FIG. 3 (commercial Na-BEA) origin)Example 12 uncalcined zeolite beta RPA70 (Drosophila melanogaster DNAbinding activity present (high) see FIG. 3 (commercial Na-BEA) origin)Example 13 uncalcined zeolite beta RPA70 (Drosophila melanogaster DNAbinding activity present (high): 100% see FIG. 3 (commercial Na-BEA)origin) Example 14 uncalcined zeolite beta RPA70 (Drosophilamelanogaster DNA binding activity present (high): 100% see FIG. 3(commercial Na-BEA) origin) Example 15 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (high): ca. 100%(commercial Na-BEA) origin) Example 16 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (medium): 49.3%(commercial Na-BEA) origin) Example 17 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (medium): 64.7%(commercial Na-BEA) origin) Example 18 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (medium): 64.4%(commercial Na-BEA) origin) Example 19 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (medium): 39.2%(commercial Na-BEA) origin) Example 20 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (high): 72.2-77.4%(commercial Na-BEA) origin) Example 21 uncalcined zeolite beta RPA70(Drosophila melanogaster DNA binding activity present (low): 24.3%(commercial Na-BEA) origin) Example 22 uncalcined zeolite beta P53(human origin) DNA binding activity present (high) the P53 used wassynthesized and (commercial Na-BEA) precipitated in E. coli Example 23uncalcined zeolite beta DNA polymerase α-catalytic subunit corepolymerase activity: 1866 DPM for a 1 hr reaction time, 3938 DPM thematerial used was synthesized and (commercial Na-BEA) domain (mouseorigin) for a 2 hr reaction time precipitated in E. coli, MW 110 kDaExample 24 uncalcined zeolite beta denatured DNA polymerase β (ratpolymerase activity: 1128000 CPM for a 1 hr reaction time the solublematerial synthesized in E. coli (commercial Na-BEA) origin) (MW 39 kDa)was denatured (polymerase activity: 0 CPM) with 6 M guanidinehydrochloride and 20 mM 2- mercaptoethanol Example 25 uncalcined zeolitebeta commercial denatured lysozyme protein lysozyme activity (units):24.5 denatured with 6 M guanidine (commercial Na-BEA) (chicken egg whiteorigin) hydrochloride and 20 mM 2-mercaptoethanol (activity: 0 units),MW 14 kDa Example 26 uncalcined zeolite RPA70 (Drosophila melanogasterDNA binding activity present (high) zeolite beta as synthesized beta(synthesized inhouse) origin) (uncalcined; only water washed and dried)Example 27 calcined zeolite beta RPA70 (Drosophila melanogaster DNAbinding activity present (low) obtained by calcination of syntheticorigin) zeolite beta; conditions: 300° C./10 hr Example 28 ″ RPA70(Drosophila melanogaster DNA binding activity present (low) obtained bycalcination of synthetic origin) zeolite beta; conditions: 400° C./8 hrExample 29 ″ RPA70 (Drosophila melanogaster DNA binding activity present(low) obtained by calcination of synthetic origin) zeolite beta;conditions: 450° C./6 hr Example 30 ″ RPA70 (Drosophila melanogaster DNAbinding activity present (low) obtained by calcination of syntheticorigin) zeolite beta; conditions: 500° C./3 hr Comp. Ex. 1 zeolite K-LTLRPA70 (Drosophila melanogaster DNA binding activity absent origin) Comp.Ex. 2 zeolite H-Y RPA70 (Drosophila melanogaster DNA binding activityabsent origin) Comp. Ex. 3 zeolite H-USY330 RPA70 (Drosophilamelanogaster DNA binding activity absent origin) Comp. Ex. 4 zeoliteH-USY360 RPA70 (Drosophila melanogaster DNA binding activity absentorigin) Comp. Ex. 5 zeolite K-FER RPA70 (Drosophila melanogaster DNAbinding activity absent origin) Comp. Ex. 7 Na-LSX RPA70 (Drosophilamelanogaster DNA binding activity absent Na-LSX: low-silica zeolite Xorigin) Comp. Ex. 8 RUB-15 RPA70 (Drosophila melanogaster DNA bindingactivity absent origin) Comp. Ex. 9 Na-FAU RPA70 (Drosophilamelanogaster DNA binding activity absent origin) Comp. Ex. 10 kanemite9RPA70 (Drosophila melanogaster DNA binding activity absent origin) Comp.Ex. 11 HOM (7 nm pore) RPA70 (Drosophila melanogaster DNA bindingactivity absent HOM: a type of mesoporous silicate origin) Comp. Ex. 12HOM (5 nm pore) RPA70 (Drosophila melanogaster DNA binding activityabsent origin) Comp. Ex. 13 HOM (6 nm pore) RPA70 (Drosophilamelanogaster DNA binding activity absent origin) Comp. Ex. 14 PLS RPA70(Drosophila melanogaster DNA binding activity absent PLS: a type oflayered silicate origin) Comp. Ex. 15 MCM-22 RPA70 (Drosophilamelanogaster DNA binding activity absent a type of layered zeoliteorigin)

TABLE 2 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Example 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 2 50 mM HEPES pH7.5/0.2M NaCl/20 mM2-mercaptoethanol/no refolding factor/1 (v/v) % Tween 20 Example 3 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 4 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 5 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v)% Triton X-100 Example 6 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % NP-40 Example 7 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/0.5(v/v) % Tween 20 Example 8 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/2 (v/v) % Tween 20 Example 9 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/3 (v/v)% Tween 20 Example 10 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/5 (v/v) % Tween 20 Example 11 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/10 (w/v) % PEG8000/1 (v/v)% Tween 20 Example 12 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/5.0 (w/v) % PEG8000/1 (v/v) % Tween 20 Example 13 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0 (w/v) % PEG8000/1(v/v) % Tween 20 Example 14 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG8000/1 (v/v) % Tween 20 Example 15 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0 (w/v) % PEG3350/1(v/v) % Tween 20 Example 16 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/10.0 (w/v) % PEG200/1 (v/v) % Tween 20 Example 17 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Example 18 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0 (w/v) % PPG2000/1 (v/v) % Tween 20 Example 19 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/5.0 (w/v) % PPG400/1(v/v) % Tween 20 Example 20 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0-5.0% Ficol170/1 (v/v) % Tween 20 Example 21 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.1% β-cyclodextrin/1(v/v) % Tween 20 Example 22 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 23 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 24 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 25 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 26 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/2 (v/v) % Tween 20 Example 27 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 28 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 29 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 30 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Comp. Ex. 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Comp. Ex. 2 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 3 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 4 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 5 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 7 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 8 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 9 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 10 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 11 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 12 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 13 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0. (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 14 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 15 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/2 (v/v) % Tween 20

Examples and Comparative Examples for the Second Aspect of the PresentInvention

These examples provided below describe the activation of a function ofprotein produced by an Escherichia coli expression system and denaturedprotein; however, the present invention is neither limited to norrestricted by these examples.

1) Preparation of Materials (a) The Refolding Agent

The following were used as the refolding agent (function activator)according to the present invention, as shown in Tables 3 and 4 below:commercially available zeolite beta, synthetic zeolite beta, calcinedzeolite beta obtained by calcination of the preceding, zeolite betacontaining various ammonium species, framework-substituted zeolite beta,and framework-substituted zeolite beta containing various ammoniumspecies. The substances listed for the comparative examples in Table 5were used as comparative products. These substances were not zeoliteswith the beta structure. For example, the zeolites in ComparativeExamples 19 and 20 were silica and silica·alumina in which the BEAstructure had yet to form due to the use of an inadequate synthesis timeand were mainly amorphous structures.

(b) The Denatured Protein Solutions

The proteins used as the target protein for activation are describedunder “protein” and “remarks” in Tables 3 to 5 and included RP70(Drosophila melanogaster origin) and P53 (human origin).

(c) The Refolding Buffer

A solution with the composition 50 mM HEPES pH 7.5/0.5 M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) polyethylene glycol 20000 (refoldingfactor)/1% (v/v) Tween 20 (detergent) was generally used as therefolding buffer. The details of the refolding buffers used are shown inTables 6 to 8.

2) The Refolding Protocol

100 mg function activator was introduced into a 1.5 mL Eppendorf tubefollowed by the addition of 0.5 mL 6 M guanidine hydrochloride·20 mMtrisaminomethane trihydrochloride (TrisHCl) pH 7.5·0.5 M NaCl 20 mM2-mercaptoethanol and suspension. To this was then added 6 M guanidinehydrochloride·20 mM 2-mercaptoethanol followed by holding for one houron ice, after which 0.5 mL of the denatured protein solution(concentration from 0.5 to 1.0 mg/mL) was added. In order to ensureadsorption of the protein on the function activator, this mixture wasstirred for 1 hour with a Rotary Culture RCC-100 (Iwaki Glass Co., Ltd.)placed in a cold room.

The function activator was then sedimented by centrifugation for 5seconds at 10000×g and the supernatant was removed. In order tocompletely remove the protein denaturant from the sedimented functionactivator, it was washed 4 times with 1 mL 20 mM TrisHCl pH 7.5·20 mM2-mercaptoethanol followed by centrifugation for 5 seconds at 10000×gand discard of the supernatant thereby produced. The remaining functionactivator was suspended by the addition thereto of 1 mL refolding buffer(comprising 50 mM HEPES pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol,refolding factor, and nonionic detergent).

In order to desorb and elute the protein adsorbed on the functionactivator, this suspension was again stirred in the cold with the RotaryCulture RCC-100 (Iwaki Glass Co., Ltd.). The function activator wasthereafter sedimented by centrifugation for 5 seconds at 10000×g, andthe protein-containing supernatant was transferred to a new Eppendorftube and this was used for activity measurement (assay).

Methods appropriate to the action of the protein used were employed forthe activity measurements. Specifically, the activity was measured usingfour types of measurements, i.e., a gel shift assay, a polymerase assay,lysozyme activity measurement, and measurement of the topoisomerase Iactivity.

3) Activity Measurement Protocols (a) The Gel Shift Assay

1 pmol radioisotope-labeled DNA oligonucleotide and the refolded proteinwere incubated for 30 minutes on ice in a solution with a composition of25 mM HEPES pH 7.4·50 mM KCl·20% glycerol·0.1% NP-40·1 mM DTT·1 mg/mLbovine serum albumin. This was followed by electrophoresis at 4° C. on4.5% polyacrylamino gel using 0.5×TBE buffer.

When DNA binding to the protein was present (that is, when activity waspresent), the protein underwent binding to the DNA, which slowed downelectrophoresis and caused band shifting, thereby enabling adetermination of activity (that is, the refolding rate).

(b) The Polymerase Assay

Poly(dA)oligo(dT)₁₂₋₁₈ or DNase I-activated calf thymus DNA was used asthe template DNA. The reaction solution had a composition (finalconcentration) of 50 mM TrisHCl pH 7.5·1 mM DTT·15% glycerol·5 mMMgCl₂·0.5 μM dTTP (cold) (thymidine triphosphate)·[3H]-dTTP (5mCi/mL:100-500 cpm/pmol). The protein (enzyme) sample solution was firstadded to and suspended in 10 μL reaction solution that was twice asconcentrated as that given above followed by incubation for 1 hour at37° C., after which the reaction was stopped by holding on ice.

The reaction solution was then dripped onto DE81 paper that had been cutinto a square. After drying, this was transferred to a beaker and waswashed in order to dissolve and remove the unreacted dTTP. This washconsisted of first 3× with 5% aqueous disodium hydrogen phosphatesolution, then 3× with distilled water, then 2× with ethanol, and wasfollowed by drying. The dry DE81 paper obtained in this manner wasplaced in a scintillator-containing vial and the radioactivity (cpm) wasmeasured with a scintillation counter. Stronger activity by the enzymesample resulted in greater incorporation of radioisotope-labeled dTTP inthe thereby synthesized DNA and thus in greater radioactivity, and theprotein activity was determined on this basis.

(c) Measurement of Lysozyme Activity

The bacteria M. lysodeikticus was selected as the substrate and wassuspended in 50 mM phosphate buffer to prepare a substrate solution witha concentration of 0.16 mg/mL. 20 μL of the protein (lysozyme enzyme)solution was added to 480 μL of this substrate solution followed byincubation for 30 minutes at room temperature. This was followed bymeasurement of the absorbance at a wavelength of 450 nm.

Lysozyme has the capacity to degrade the cell wall of bacteria, and as aresult the higher this capacity, that is, the activity, the greater thedecline in absorbance. 1 unit of lysozyme activity was defined as adecline in absorbance at 450 nm of 0.001 per minute.

(d) Measurement of the Topoisomerase I Activity

0.5 μg supercoiled pBR322 and the topoisomerase I protein were suspendedin reaction buffer (10 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mMbeta-mercaptoethanol, 0.5 mM EDTA). After incubation for 30 minutes at37° C., 0.1% SDS was added to stop the reaction. 0.5 μg/mL proteinase Kwas then added followed by incubation for 30 minutes at 37° C. in orderto degrade the topoisomerase I protein in the solution. The solution wassubsequently subjected to electrophoresis on 1% (w/v) agarose and theDNA was stained with 0.5 μg/mL ethidium bromide. The topoisomerase Iactivity was measured based on observation of an upward shifted DNA bandwith a UV trans illuminator.

The activity (refolding rate) and protein recovery rate, which are theresults for the subject examples obtained by the procedures andprotocols described above, are shown in Tables 3 and 4 while the resultsfor the comparative examples are shown in Table 5. As shown by theexamples, activity native to the proteins, for example, DNA bindingactivity, is produced by refolding. The zeolite beta according to thepresent invention is useful as a highly versatile, highly generalizablerefolding agent that is applicable to a variety of denatured anddeactivated proteins and proteins that have an as yet unformed higherorder structure; however, the application thereof is not limited to theproteins shown in the examples and the zeolite beta according to thepresent invention can be applied to any protein.

TABLE 3 function activator protein activity (refolding rate); proteinrecovery rate remarks Example 1 uncalcined zeolite RPA70 (Drosophila DNAbinding activity present (high) the RPA70 used was synthesized beta(BEA: contains melanogaster origin) and precipitated in E. coli, MW theamine TEA) 66 kDa Example 2 uncalcined zeolite RPA70 (Drosophila DNAbinding activity present (medium) beta (BEA: contains melanogasterorigin) the amine TEA) Example 3 uncalcined zeolite RPA70 (DrosophilaDNA binding activity present (low) beta (BEA: contains melanogasterorigin) the amine TEA) Example 4 uncalcined zeolite RPA70 (DrosophilaDNA binding activity present (high): ca. 90%; 20% beta (BEA: containsmelanogaster origin) the amine TEA) Example 5 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high) beta (BEA: containsmelanogaster origin) the amine TEA) Example 6 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high) beta (BEA: containsmelanogaster origin) the amine TEA) Example 7 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high): ca. 80%; 16% beta (BEA:contains melanogaster origin) the amine TEA) Example 8 uncalcinedzeolite RPA70 (Drosophila DNA binding activity present (high): a littleover 95%; beta (BEA: contains melanogaster origin) 23% the amine TEA)Example 9 uncalcined zeolite RPA70 (Drosophila DNA binding activitypresent (high): ca. 95%; 22% beta (BEA: contains melanogaster origin)the amine TEA) Example 10 uncalcined zeolite RPA70 (Drosophila DNAbinding activity present (high): a little over 90%; beta (BEA: containsmelanogaster origin) 19% the amine TEA) Example 11 uncalcined zeoliteRPA70 (Drosophila DNA binding activity present (low) beta (BEA: containsmelanogaster origin) the amine TEA) Example 12 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high) beta (BEA: containsmelanogaster origin) the amine TEA) Example 13 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high): 100% beta (BEA:contains melanogaster origin) the amine TEA) Example 14 uncalcinedzeolite RPA70 (Drosophila DNA binding activity present (high): 100% beta(BEA: contains melanogaster origin) the amine TEA) Example 15 uncalcinedzeolite RPA70 (Drosophila DNA binding activity present (high): ca. 100%beta (BEA: contains melanogaster origin) the amine TEA) Example 16uncalcined zeolite RPA70 (Drosophila DNA binding activity present(medium): 49.3% beta (BEA: contains melanogaster origin) the amine TEA)Example 17 uncalcined zeolite RPA70 (Drosophila DNA binding activitypresent (medium): 64.7% beta (BEA: contains melanogaster origin) theamine TEA) Example 18 uncalcined zeolite RPA70 (Drosophila DNA bindingactivity present (medium): 64.4% beta (BEA: contains melanogasterorigin) the amine TEA) Example 19 uncalcined zeolite RPA70 (DrosophilaDNA binding activity present (medium): 39.2% beta (BEA: containsmelanogaster origin) the amine TEA) Example 20 uncalcined zeolite RPA70(Drosophila DNA binding activity present (high): 72.2-77.4% beta (BEA:contains melanogaster origin) the amine TEA) Example 21 uncalcinedzeolite RPA70 (Drosophila DNA binding activity present (low): 24.3% beta(BEA: contains melanogaster origin) the amine TEA) Example 22 uncalcinedzeolite P53 (human origin) DNA binding activity present (high) the P53used was synthesized and beta (BEA: contains precipitated in E. coli theamine TEA) Example 23 uncalcined zeolite DNA polymerase α · polymeraseactivity: 1866DPM for a 1 hr reaction the material used was synthesizedbeta (BEA: contains catalytic subunit core time, 3938DPM for a 2 hrreaction time and precipitated in E. coli, MW the amine TEA) domain(mouse origin) 110 kDa Example 24 uncalcined zeolite denatured DNApolymerase activity: 1128000 for a 1 hr reaction time the solublematerial synthesized beta (BEA: contains polymerase β (rat in E. coli(MW 39 kDa) was the amine TEA) origin) denatured (polymerase activity: 0CPM) with 6 M guanidine hydrochloride and 20 mM 2- mercaptoethanolExample 25 uncalcined zeolite commercial denatured lysozyme activity(units): 24.5 denatured with 6 M guanidine beta (BEA: contains lysozymeprotein hydrochloride and 20 mM 2- the amine TEA) (chicken egg whitemercapto-ethanol (activity: 0 origin) units), MW 14 kDa

TABLE 4 protein recovery function activator protein activity (refoldingrate); rate remarks Example 26 calcined zeolite beta RPA70 (DrosophilaDNA binding activity present (low) calcination conditions: 300° C./10 hrmelanogaster origin) Example 27 ″ RPA70 (Drosophila DNA binding activitypresent (low) calcination conditions: 400° C./8 hr melanogaster origin)Example 28 ″ RPA70 (Drosophila DNA binding activity present (low)calcination conditions: 450° C./6 hr melanogaster origin) Example 29 ″RPA70 (Drosophila DNA binding activity present (low) calcinationconditions: 500° C./3 hr melanogaster origin) Example 30 uncalcinedzeolite beta RPA70 (Drosophila DNA binding activity present (high) (BEA:contains the melanogaster origin) amine TEA) Example 31 uncalcinedzeolite beta DNA polymerase δ polymerase activity: 16641DPM for asynthesized and precipitated in (BEA: contains the (rice origin) amino 1hr reaction time E. coli, MW 120 kDa, activity amine TEA) acid sequence1-50 measured by the same method as deleted in Example 24 Example 32zeolite beta RPA70 (Drosophila DNA binding activity present (low) (notemplate: no melanogaster origin) amine) Example 33 zeolite beta RPA70(Drosophila DNA binding activity present (low) (amine: TBA) melanogasterorigin) Example 34 zeolite beta RPA70 (Drosophila DNA binding activitypresent (high) (amine: TMA) melanogaster origin) Example 35 zeolite betaRPA70 (Drosophila DNA binding activity present (high) (amine: TEA)melanogaster origin) Example 36 zeolite beta RPA70 (Drosophila DNAbinding activity present (high) (amine: TPA) melanogaster origin)Example 37 zeolite beta RPA70 (Drosophila DNA binding activity present(low) (amine: pyridine) melanogaster origin) Example 26 calcined zeolitebeta RPA70 (Drosophila DNA binding activity present (low) calcinationconditions: 300° C./10 hr melanogaster origin) Example 27 ″ RPA70(Drosophila DNA binding activity present (low) calcination conditions:400° C./8 hr melanogaster origin) Example 28 ″ RPA70 (Drosophila DNAbinding activity present (low) calcination conditions: 450° C./6 hrmelanogaster origin) Example 29 ″ RPA70 (Drosophila DNA binding activitypresent (low) calcination conditions: 500° C./3 hr melanogaster origin)Example 30 uncalcined zeolite beta RPA70 (Drosophila DNA bindingactivity present (high) (BEA: contains the melanogaster origin) amineTEA) Example 31 uncalcined zeolite beta DNA polymerase δ polymeraseactivity: 16641DPM for a synthesized and precipitated in (BEA: containsthe (rice origin) amino 1 hr reaction time E. coli, MW 120 kDa, activityamine TEA) acid sequence 1-50 measured by the same method as deleted inExample 24 Example 32 zeolite beta (no RPA70 (Drosophila DNA bindingactivity present (low) template: no amine) melanogaster origin) Example33 zeolite beta RPA70 (Drosophila DNA binding activity present (low)(amine: TBA) melanogaster origin) Example 34 zeolite beta RPA70(Drosophila DNA binding activity present (high) (amine: TMA)melanogaster origin) Example 35 zeolite beta RPA70 (Drosophila DNAbinding activity present (high) (amine: TEA) melanogaster origin)Example 36 zeolite beta RPA70 (Drosophila DNA binding activity present(high) (amine: TPA) melanogaster origin) Example 37 zeolite beta RPA70(Drosophila DNA binding activity present (low) (amine: pyridine)melanogaster origin) Example 38 Si-rich BEA RPA70 (Drosophila DNAbinding activity present (low) melanogaster origin) Example 39 Al-richBEA RPA70 (Drosophila DNA binding activity present (low) melanogasterorigin) Example 40 Co BEA RPA70 (Drosophila DNA binding activity present(low) melanogaster origin) Example 41 Ti BEA RPA70 (Drosophila DNAbinding activity present (low) melanogaster origin) Example 42BEA-ammonia RPA70 (Drosophila DNA binding activity present (low)melanogaster origin) Example 43 BEA-urea RPA70 (Drosophila DNA bindingactivity present (low) melanogaster origin) Example 44 NaBEA, 135° C.,RPA70 (Drosophila DNA binding activity present (low) 27 hr melanogasterorigin) Example 45 NaBEA, 96 hr RPA70 (Drosophila DNA binding activitypresent (low) melanogaster origin) Example 46 NaBEA Topoisomerase I DNArelaxation activity present MW 112 kDa. Material recovered (Drosophila(high): 89% with the soluble fraction from E. coli melanogaster origin)was used for the activity comparison. The refolded material was thematerial synthesized and precipitated in E. coli.

TABLE 5 activity (refolding rate); function activator protein proteinrecovery rate remarks Comp. Ex. 1 zeolite K-LTL RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 2 zeolite H-YRPA70 (Drosophila DNA binding activity absent melanogaster origin) Comp.Ex. 3 zeolite H-USY330 RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 4 zeolite H-USY360 RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 5 zeolite K-FERRPA70 (Drosophila DNA binding activity absent melanogaster origin) Comp.Ex. 7 Na-LSX RPA70 (Drosophila DNA binding activity absent Na-LSX:low-silica zeolite X melanogaster origin) Comp. Ex. 8 RUB-15 RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex. 9Na-FAU RPA70 (Drosophila DNA binding activity absent melanogasterorigin) Comp. Ex. 10 kanemite9 RPA70 (Drosophila DNA binding activityabsent melanogaster origin) Comp. Ex. 11 HOM (pore 7 nm) RPA70(Drosophila DNA binding activity absent HOM: a type of mesoporoussilicate melanogaster origin) Comp. Ex. 12 HOM (pore 5 nm) RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex.13 HOM (pore 6 nm) RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 14 PLS RPA70 (Drosophila DNA bindingactivity absent PLS: a type of layered silicate melanogaster origin)Comp. Ex. 15 MCM-22 RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 16 FER-TEA RPA70 (Drosophila DNA bindingactivity absent melanogaster origin) Comp. Ex. 17 MOR-TEA RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex.18 FER-pyridine RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 19 silica with a not yet RPA70(Drosophila DNA binding activity absent formed zeolite beta melanogasterorigin) structure (BEA 135° C., 24 hr) Comp. Ex. 20 GaBEA RPA70(Drosophila DNA binding activity absent melanogaster origin)

TABLE 6 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Example 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 2 50 mM HEPES pH7.5/0.2M NaCl/20 mM2-mercaptoethanol/no refolding factor/1 (v/v) % Tween 20 Example 3 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 4 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 5 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v)% Triton X-100 Example 6 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % NP-40 Example 7 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/0.5(v/v) % Tween 20 Example 8 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/2 (v/v) % Tween 20 Example 9 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/3 (v/v)% Tween 20 Example 10 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/5 (v/v) % Tween 20 Example 11 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/10% (w/v) PEG8000/1 (v/v)% Tween 20 Example 12 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/5.0% (w/v) PEG8000/1 (v/v) % Tween 20 Example 13 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0% (w/v) PEG8000/1 (v/v)% Tween 20 Example 14 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG8000/1 (v/v) % Tween 20 Example 15 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0% (w/v) PEG3350/1 (v/v)% Tween 20 Example 16 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/10.0% (w/v) PEG200/1 (v/v) % Tween 20 Example 17 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 18 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0% (w/v) PPG2000/1 (v/v) % Tween 20 Example 19 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/5.0% (w/v) PPG400/1 (v/v)% Tween 20 Example 20 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0-5.0% Ficol170/1 (v/v) % Tween 20 Example 21 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.1% β-cyclodextrin/1(v/v) % Tween 20 Example 22 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 23 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 24 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 25 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20

TABLE 7 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Example 26 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 27 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 28 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 29 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 30 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/2 (v/v)% Tween 20 Example 31 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 32 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 33 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 34 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 35 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 36 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 37 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 38 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 39 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 40 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 41 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 42 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 43 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 44 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 45 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 46 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20

TABLE 8 refolding buffer 50 mM HEPES pH7.5/0.5 MNaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Comp. Ex. 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Comp. Ex. 2 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 3 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 4 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 5 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 7 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 8 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 9 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 10 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 11 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 12 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 13 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 14 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 15 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/2 (v/v) % Tween 20 Comp. Ex. 16 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 17 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 18 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 19 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 20 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20

Examples and Comparative Examples for the Third Aspect of the PresentInvention

These examples provided below describe the activation of a function ofprotein produced by an Escherichia coli expression system and denaturedprotein; however, the present invention is neither limited to norrestricted by these examples.

1) Preparation of Materials (a) The Refolding Agent

The following were used as the refolding agent (function activator), asshown in Tables 9 and 10 below: commercially available zeolite beta,synthetic zeolite beta, calcined zeolite beta obtained by calcination ofthe preceding, zeolite beta containing various ammonium species,framework-substituted zeolite beta, and framework-substituted zeolitebeta containing various ammonium species. The substances listed for thecomparative examples in Table 13 were used as comparative products.These substances were not zeolites with the beta structure. For example,the substances in Comparative Examples 19 and 20 were silica andsilica·alumina in which the BEA structure had yet to form due to the useof an inadequate synthesis time and were mainly amorphous structures.

(b) The Denatured Protein Solutions

The proteins described in Tables 9 and 10, RP70 (Drosophila melanogasterorigin), P53 (human origin), and so forth were used as the targetproteins for activation.

(c) The Refolding Buffer

A solution with the composition 50 mM HEPES pH 7.5/0.5 M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) polyethylene glycol 20000 (refoldingfactor)/1% (v/v) Tween 20 (detergent) was generally used as therefolding buffer. The details of the refolding buffers used are shown inTables 11, 12, and 14.

2) The Refolding Protocol

100 mg refolding agent was introduced into a 1.5 mL Eppendorf tubefollowed by the addition of 0.5 mL 6 M guanidine hydrochloride·20 mMtrisaminomethane trihydrochloride (TrisHCl) pH 7.5·0.5 M NaCl·20 mM2-mercaptoethanol and suspension. To this was then added 6 M guanidinehydrochloride·20 mM 2-mercaptoethanol followed by holding for one houron ice, after which 0.5 mL of the denatured protein solution(concentration from 0.5 to 1.0 mg/mL) was added. In order to ensureadsorption of the protein on the refolding agent, this mixture wasstirred for 1 hour with a rotary culture unit (Rotary Culture RCC-100from Iwaki Glass Co., Ltd.) placed in a cold room.

The refolding agent was then sedimented by centrifugation for 5 secondsat 10000×g and the supernatant was removed. In order to completelyremove the protein denaturant from the sedimented refolding agent, itwas washed 4 times with 1 mL 20 mM TrisHCl pH 7.5·20 mM2-mercaptoethanol followed by centrifugation for 5 seconds at 10000×gand discard of the supernatant thereby produced. The remaining refoldingagent was suspended by the addition thereto of 1 mL refolding buffer(comprising 50 mM HEPES pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol,refolding factor, and nonionic detergent).

In order to desorb and elute the protein adsorbed on the refoldingagent, this suspension was again stirred in the cold with a rotaryculture unit (Rotary Culture RCC-100, Iwaki Glass Co., Ltd.). Therefolding agent was thereafter sedimented by centrifugation for 5seconds at 10000×g, and the protein-containing supernatant wastransferred to a new Eppendorf tube and this was used for activitymeasurement (assay).

Methods appropriate to the action of the protein used were employed forthe activity measurements. Specifically, the activity was measured usingfour types of measurements, i.e., a gel shift assay, a polymerase assay,lysozyme activity measurement, and measurement of the topoisomerase Iactivity.

3) Activity Measurement Protocols (a) The Gel Shift Assay

1 pmol radioisotope-labeled DNA oligonucleotide and the refolded proteinwere incubated for 30 minutes on ice in a solution with a composition of25 mM HEPES pH 7.4·50 mM KCl·20% glycerol·0.1% NP-40·1 mM DTT·1 mg/mLbovine serum albumin. This was followed by electrophoresis at 4° C. on4.5% polyacrylamino gel using 0.5×TBE buffer.

When DNA binding to the protein was present (that is, when activity waspresent), the protein underwent binding to the DNA, which slowed downelectrophoresis and caused band shifting, thereby enabling adetermination of activity (that is, the refolding rate).

(b) The Polymerase Assay

Poly(dA)oligo(dT)₁₂₋₁₈ or DNase I-activated calf thymus DNA was used asthe template DNA. The reaction solution had a composition (finalconcentration) of 50 mM TrisHCl pH 7.5·1 mM DTT·15% glycerol·5 mMMgCl₂·0.5 μM dTTP (cold) (thymidine triphosphate)·[³H]-dTTP (5mCi/mL:100-500 cpm/pmol). The protein (enzyme) sample solution was firstadded to and suspended in 10 μL reaction solution that was twice asconcentrated as that given above followed by incubation for 1 hour at37° C., after which the reaction was stopped by holding on ice.

The reaction solution was then dripped onto DE81 paper that had been cutinto a square. After drying, this was transferred to a beaker and waswashed in order to dissolve and remove the unreacted dTTP. This washconsisted of first 3× with 5% aqueous disodium hydrogen phosphatesolution, then 3× with distilled water, then 2× with ethanol, and wasfollowed by drying. The dry DE81 paper obtained in this manner wasplaced in a scintillator-containing vial and the radioactivity (cpm) wasmeasured with a scintillation counter. Stronger activity by the enzymesample resulted in greater incorporation of radioisotope-labeled dTTP inthe thereby synthesized DNA and thus in greater radioactivity, and theprotein activity was determined on this basis.

(c) Measurement of Lysozyme Activity

The bacteria M. lysodeikticus was selected as the substrate and wassuspended in 50 mM phosphate buffer to prepare a substrate solution witha concentration of 0.16 mg/mL. 20 μL of the protein (lysozyme enzyme)solution was added to 480 μL of this substrate solution followed byincubation for 30 minutes at room temperature. This was followed bymeasurement of the absorbance at a wavelength of 450 nm.

Lysozyme has the capacity to degrade the cell wall of bacteria, and as aresult the higher this capacity, that is, the activity, the greater thedecline in absorbance. 1 unit of lysozyme activity was defined as adecline in absorbance at 450 nm of 0.001 per minute.

(d) Measurement of the Topoisomerase I Activity

0.5 μg supercoiled pBR322 and the topoisomerase I protein were suspendedin reaction buffer (10 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mMbeta-mercaptoethanol, 0.5 mM EDTA). After incubation for 30 minutes at37° C., 0.1% SDS was added to stop the reaction. 0.5 μg/mL proteinase Kwas then added followed by incubation for 30 minutes at 37° C. in orderto degrade the topoisomerase I protein in the solution. The solution wassubsequently subjected to electrophoresis on 1% (w/v) agarose and theDNA was stained with 0.5 μg/mL ethidium bromide. The topoisomerase Iactivity was measured based on observation of an upward shifted DNA bandwith a UV trans illuminator.

The activity (refolding rate) and protein recovery rate, which are theresults for the subject examples obtained by the procedures andprotocols described above, are shown in Tables 9, 10, and 13 togetherwith the results for the comparative examples. As shown by the examples,activity native to the proteins, for example, the DNA binding activity,is produced by refolding. The refolding agent according to the presentinvention is useful as a highly versatile, highly generalizablerefolding agent that is applicable to a variety of denatured anddeactivated proteins and proteins that have an as yet unformed higherorder structure; however, the application thereof is not limited to theproteins shown in the examples and the refolding agent according to thepresent invention can be applied to any protein.

TABLE 9 function activator protein Example 1 uncalcined zeolite beta(BEA: contains the amine TEA) RPA70 (Drosophila melanogaster origin)Example 2 ″ ″ Example 3 ″ ″ Example 4 ″ ″ Example 5 ″ ″ Example 6 ″ ″Example 7 ″ ″ Example 8 ″ ″ Example 9 ″ ″ Example 10 ″ ″ Example 11 ″ ″Example 12 ″ ″ Example 13 ″ ″ Example 14 ″ ″ Example 15 ″ ″ Example 16 ″″ Example 17 ″ ″ Example 18 ″ ″ Example 19 ″ ″ Example 20 ″ ″ Example 21″ ″ Example 22 ″ P53 (human origin) Example 23 ″ DNA polymerase α″catalytic subunit core domain (mouse origin) Example 24 ″ denatured DNApolymerase β (rat origin) Example 25 ″ commercial denatured lysozymeprotein (chicken egg white origin) Example 26 calcined zeolite betaRPA70 (Drosophila melanogaster origin) Example 27 ″ ″ Example 28 ″ ″Example 29 ″ ″ Example 30 uncalcined zeolite beta (BEA: ″ contains theamine TEA) activity (refolding rate); protein recovery rate remarksExample 1 DNA binding activity present (high) the RPA70 used wassynthesized and precipitated in E. coli, MW 66 kDa Example 2 DNA bindingactivity present (medium) Example 3 DNA binding activity present (low)Example 4 DNA binding activity present (high): ca. 90%; 20% Example 5DNA binding activity present (high) Example 6 DNA binding activitypresent (high) Example 7 DNA binding activity present (high): ca. 80%;16% Example 8 DNA binding activity present (high): a little over 95%;23% Example 9 DNA binding activity present (high): ca. 95%; 22% Example10 DNA binding activity present (high): a little over 90%; 19% Example11 DNA binding activity present (low) Example 12 DNA binding activitypresent (high) Example 13 DNA binding activity present (high): 100%Example 14 DNA binding activity present (high): 100% Example 15 DNAbinding activity present (high): ca. 100% Example 16 DNA bindingactivity present (medium): 49.3% Example 17 DNA binding activity present(medium): 64.7% Example 18 DNA binding activity present (medium): 64.4%Example 19 DNA binding activity present (medium): 39.2% Example 20 DNAbinding activity present (high): 72.2-77.4% Example 21 DNA bindingactivity present (low): 24.3% Example 22 DNA binding activity present(high) the P53 used was synthesized and precipitated in E. coli Example23 polymerase activity: 1866 DPM for a 1 hr reaction time, 3938 thematerial used was synthesized and DPM for a 2 hr reaction timeprecipitated in E. coli, MW 110 kDa Example 24 polymerase activity:1128000 CPM for a 1 hr reaction time the soluble material synthesized inE. coli (MW 39 kDa) was denatured (polymerase activity: 0 CPM) with 6Mguanidine hydrochloride and 20 mM 2-mercaptoethanol Example 25 lysozymeactivity (units): 24.5 denatured with 6M guanidine hydrochloride and 20mM 2-mercapto-ethanol (activity: 0 units): MW 14 kDa Example 26 DNAbinding activity present (low) calcination conditions: 300° C./10 hrExample 27 DNA binding activity present (low) calcination conditions:400° C./8 hr Example 28 DNA binding activity present (low) calcinationconditions: 450° C./6 hr Example 29 DNA binding activity present (low)calcination conditions: 500° C./3 hr Example 30 DNA binding activitypresent (high)

TABLE 10 function activator protein Example 31 as same as the above DNApolymerase δ (rice origin) amino acid sequence 1-50 deleted Example 32zeolite beta (no template: no amine) RPA70 (Drosophila melanogasterorigin) Example 33 zeolite beta (amine: TBA) ″ Example 34 zeolite beta(amine: TMA) ″ Example 35 zeolite beta (amine: TEA) ″ Example 36 zeolitebeta (amine: TPA) ″ Example 37 zeolite beta (amine: pyridine) ″ Example38 Si-rich BEA RPA70 (Drosophila melanogaster origin) Example 39 Al-richBEA ″ Example 40 Co BEA ″ Example 41 Ti BEA ″ Example 42 BEA-ammonia ″Example 43 BEA-urea ″ Example 44 NaBEA, 135° C., 27 hr ″ Example 45NaBEA, 96 hr ″ Example 46 NaBEA Topoisomerase I (Drosophila melanogasterorigin) Example 47 zeolite beta (amine: monomethyl) RPA70 (Drosophilamelanogaster origin) Example 48 zeolite beta (amine: dimethyl) ″ Example49 zeolite beta (amine: trimethyl) ″ Example 50 zeolite beta (amine:monoethyl) ″ Example 51 zeolite beta (amine: diethyl) ″ Example 52zeolite beta (amine: triethyl) ″ Example 53 zeolite beta (amine:monopropyl) ″ Example 54 zeolite beta (amine: dipropyl) ″ Example 55zeolite beta (amine: tripropyl) ″ Example 56 zeolite beta (amine:monobutyl) ″ Example 57 zeolite beta (amine: dibutyl) ″ Example 58zeolite beta (amine: tributyl) ″ activity (refolding rate); proteinrecovery rate remarks Example 31 polymerase activity: 16641 DPM for a 1hr synthesized and precipitated in E. coli, reaction time MW 120 kDa,activity measured by the same method as in Example 24 Example 32 DNAbinding activity present (low) Example 33 DNA binding activity present(low) Example 34 DNA binding activity present (high) Example 35 DNAbinding activity present (high) Example 36 DNA binding activity present(high) Example 37 DNA binding activity present (low) Example 38 DNAbinding activity present (low) Example 39 DNA binding activity present(low) Example 40 DNA binding activity present (low) Example 41 DNAbinding activity present (low) Example 42 DNA binding activity present(low) Example 43 DNA binding activity present (low) Example 44 DNAbinding activity present (low) Example 45 DNA binding activity present(low) Example 46 DNA relaxation activity present (high): 89% MW 112 kD.Material recovered with the soluble fraction from E. coli was used forthe activity comparison. The refolded material was the materialsynthesized and precipitated in E. coli. Example 47 DNA binding activitypresent (low) Example 48 DNA binding activity present (medium) Example49 DNA binding activity present (medium) Example 50 DNA binding activitypresent (medium) Example 51 DNA binding activity present (medium)Example 52 DNA binding activity present (large) Example 53 DNA bindingactivity present (medium) Example 54 DNA binding activity present(large) Example 55 DNA binding activity present (large) Example 56 DNAbinding activity present (large) Example 57 DNA binding activity present(large) Example 58 DNA binding activity present (large)

TABLE 11 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Example 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 2 50 mM HEPES pH7.5/0.2M NaCl/20 mM2-mercaptoethanol/no refolding factor/1 (v/v) % Tween 20 Example 3 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/no refolding factor/1(v/v) % Tween 20 Example 4 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 5 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v)% Triton X-100 Example 6 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/1 (v/v) % NP-40 Example 7 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/0.5(v/v) % Tween 20 Example 8 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/2 (v/v) % Tween 20 Example 9 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/2.5% (w/v) PEG20K/3 (v/v)% Tween 20 Example 10 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/2.5% (w/v) PEG20K/5 (v/v) % Tween 20 Example 11 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/10 (w/v) % PEG8000/1 (v/v)% Tween 20 Example 12 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/5.0 (w/v) % PEG8000/1 (v/v) % Tween 20 Example 13 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0 (w/v) % PEG8000/1(v/v) % Tween 20 Example 14 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG8000/1 (v/v) % Tween 20 Example 15 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/1.0 (w/v) % PEG3350/1(v/v) % Tween 20 Example 16 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/10.0 (w/v) % PEG200/1 (v/v) % Tween 20 Example 17 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Example 18 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0 (w/v) % PPG2000/1 (v/v) % Tween 20 Example 19 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/5.0 (w/v) % PPG400/1(v/v) % Tween 20 Example 20 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/1.0-5.0% Ficol170/1 (v/v) % Tween 20 Example 21 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.1% β-cyclodextrin/1(v/v) % Tween 20 Example 22 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 23 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 24 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 25 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 26 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 27 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 28 50 mM HEPES pH7.5/0.1M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v) % Tween 20 Example 29 50 mMHEPES pH7.5/0.1M NaCl/20 mM 2-mercaptoethanol/0.5% (w/v) PEG20K/1 (v/v)% Tween 20 Example 30 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) PEG20K/2 (v/v) % Tween 20

TABLE 12 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Example 31 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 32 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 33 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 34 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 35 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 36 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 37 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 38 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 39 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 40 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 41 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 42 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 43 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 44 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 45 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 46 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 47 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 48 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 49 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 50 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 51 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 52 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 53 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 54 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 55 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 56 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Example 57 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Example 58 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20

TABLE 13 activity (refolding rate); function activator protein proteinrecovery rate remarks Comp. Ex. 1 zeolite K-LTL RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 2 zeolite H-YRPA70 (Drosophila DNA binding activity absent melanogaster origin) Comp.Ex. 3 zeolite H-USY330 RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 4 zeolite H-USY360 RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 5 zeolite K-FERRPA70 (Drosophila DNA binding activity absent melanogaster origin) Comp.Ex. 7 Na-LSX RPA70 (Drosophila DNA binding activity absent Na-LSX:low-silica melanogaster origin) zeolite X Comp. Ex. 8 RUB-15 RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex. 9Na-FAU RPA70 (Drosophila DNA binding activity absent melanogasterorigin) Comp. Ex. 10 kanemite9 RPA70 (Drosophila DNA binding activityabsent melanogaster origin) Comp. Ex. 11 HOM (pore 7 nm) RPA70(Drosophila DNA binding activity absent HOM: a type of melanogasterorigin) mesoporous silicate Comp. Ex. 12 HOM (pore 5 nm) RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex.13 HOM (pre 6) RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 14 PLS RPA70 (Drosophila DNA bindingactivity absent PLS: a type of melanogaster origin) layered silicateComp. Ex. 15 MCM-22 RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 16 FER-TEA RPA70 (Drosophila DNA bindingactivity absent melanogaster origin) Comp. Ex. 17 MOR-TEA RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex.18 FER-pyridine RPA70 (Drosophila DNA binding activity absentmelanogaster origin) Comp. Ex. 19 silica with a not yet formed zeoliteRPA70 (Drosophila DNA binding activity absent beta structure (BEA 135°C., melanogaster origin) 24 hr) Comp. Ex. 20 GaBEA with a not yet formedzeolite RPA70 (Drosophila DNA binding activity absent beta structuremelanogaster origin) Comp. Ex. 21 ZSM-5 (MFI-TPA) RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 22 silicalite(MFI-TPA) RPA70 (Drosophila DNA binding activity absent melanogasterorigin) Comp. Ex. 23 additional impregnation of TPA in H⁺ RPA70(Drosophila trace DNA binding activity (≦0.1%) form of Comp. Ex. 22melanogaster origin) Comp. Ex. 24 AlPO₄-5 (amine: triethyl) RPA70(Drosophila trace DNA binding activity (≦0.1%) melanogaster origin)Comp. Ex. 25 calcined AlPO₄-5 (of Comp. Ex. 23) RPA70 (Drosophila DNAbinding activity absent melanogaster origin) Comp. Ex. 26 uncalcinedCoAlPO₄-5 RPA70 (Drosophila DNA binding activity absent melanogasterorigin) Comp. Ex. 27 calcined material from Comp. Ex. 26 RPA70(Drosophila DNA binding activity absent melanogaster origin) Comp. Ex.28 SnAlPO₄-5 RPA70 (Drosophila trace DNA binding activity (≦0.1%)melanogaster origin) Comp. Ex. 29 calcined material from Comp. Ex. 28RPA70 (Drosophila DNA binding activity absent melanogaster origin)

TABLE 14 refolding buffer 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/refolding factor/nonionic detergent Comp. Ex. 1 50 mMHEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v)% Tween 20 Comp. Ex. 2 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 3 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 4 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 5 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 7 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 8 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 9 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 10 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 11 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 12 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 13 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 14 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 15 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/2 (v/v) % Tween 20 Comp. Ex. 16 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 17 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 18 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 19 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 20 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 21 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 22 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 23 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 24 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 25 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 26 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 27 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20 Comp. Ex. 28 50mM HEPES pH7.5/0.5M NaCl/20 mM 2-mercaptoethanol/0.5 (w/v) % PEG20K/1(v/v) % Tween 20 Comp. Ex. 29 50 mM HEPES pH7.5/0.5M NaCl/20 mM2-mercaptoethanol/0.5 (w/v) % PEG20K/1 (v/v) % Tween 20

Examples and Comparative Examples for the Fourth Aspect of the PresentInvention

These examples provided below describe the activation of a function ofprotein produced by an Escherichia coli expression system and denaturedprotein; however, the present invention is neither limited to norrestricted by these examples.

1) Preparation of Materials (a) The Refolding Moldings

Refolding moldings were fabricated by the following methods: conversionto zeolite beta by dry conversion and solid-phase conversion of anamorphous silica·alumina molding and an amorphous silica·alumina filmcoated on the surface of an inorganic ceramic support; deposition andimmobilization of zeolite beta by in situ synthesis on filter paper andon the surface of an inorganic ceramic support; immobilization ofzeolite beta on a support surface using an adhesive; and immobilizationof zeolite beta utilizing an adhesive surface, such as adhesive tape. Inthe latter two methods, which employ an adhesive and an adhesivesurface, the moldings were fabricated using zeolite beta that had beensynthesized inhouse in advance, or using the commercially acquiredproduct, and using various zeolite betas obtained by modifying thepreceding by, for example, ion exchange.

(b) The Denatured Protein Solutions

RPA70 (Drosophila melanogaster origin), P53 (human origin), and so forthwere used as the target proteins for activation.

(c) The Refolding Buffer

A solution with the composition 50 mM HEPES pH 7.5/0.5 M NaCl/20 mM2-mercaptoethanol/0.5% (w/v) polyethylene glycol 20000 (refoldingfactor)/1% (v/v) Tween 20 (detergent) was generally used as therefolding buffer.

2) Activity Measurement Protocols

Methods appropriate to the action of the protein used were employed forthe activity measurements. Specifically, the four types of measurementsdescribed below, i.e., a gel shift assay, a polymerase assay, lysozymeactivity measurement, and measurement of the topoisomerase I activity,were carried out.

(a) The Gel Shift Assay

1 pmol radioisotope-labeled DNA oligonucleotide and the refolded proteinwere incubated for 30 minutes on ice in a solution with a composition of25 mM HEPES pH 7.4·50 mM KCl·20% glycerol·0.1% NP-40·1 mM DTT·1 mg/mLbovine serum albumin. This was followed by electrophoresis at 4° C. on4.5% polyacrylamino gel using 0.5×TBE buffer.

When DNA binding to the protein was present (that is, when activity waspresent), the protein underwent binding to the DNA, which slowed downelectrophoresis and caused band shifting, thereby enabling adetermination of activity (that is, the refolding rate).

(b) The Polymerase Assay

Poly(dA)oligo(dT)₁₂₋₁₈ or DNase I-activated calf thymus DNA was used asthe template DNA. The reaction solution had a composition (finalconcentration) of 50 mM TrisHCl pH 7.5·1 mM DTT·15% glycerol·5 mMMgCl₂·0.5 μM dTTP (cold) (thymidine triphosphate)·[³H]-dTTP (5mCi/mL:100-500 cpm/pmol). The protein (enzyme) sample solution was firstadded to and suspended in 10 μL reaction solution that was twice asconcentrated as that given above followed by incubation for 1 hour at37° C., after which the reaction was stopped by holding on ice.

The reaction solution was then dripped onto DE81 paper that had been cutinto a square. After drying, this was transferred to a beaker and waswashed in order to dissolve and remove the unreacted dTTP. This washconsisted of first 3× with 5% aqueous disodium hydrogen phosphatesolution, then 3× with distilled water, then 2× with ethanol, and wasfollowed by drying. The dry DE81 paper obtained in this manner wasplaced in a scintillator-containing vial and the radioactivity (cpm) wasmeasured with a scintillation counter. Stronger activity by the enzymesample resulted in greater incorporation of radioisotope-labeled dTTP inthe thereby synthesized DNA and thus in greater radioactivity, and theprotein activity was determined on this basis.

(c) Measurement of Lysozyme Activity

The bacteria M. lysodeikticus was selected as the substrate and wassuspended in 50 mM phosphate buffer to prepare a substrate solution witha concentration of 0.16 mg/mL. 20 μL of the protein (lysozyme enzyme)solution was added to 480 μL of this substrate solution followed byincubation for 30 minutes at room temperature. This was followed bymeasurement of the absorbance at a wavelength of 450 nm. Lysozyme hasthe capacity to degrade the cell wall of bacteria, and as a result thehigher this capacity, that is, the activity, the greater the decline inabsorbance. 1 unit of lysozyme activity was defined as a decline inabsorbance at 450 nm of 0.001 per minute.

(d) Measurement of the Topoisomerase I Activity

0.5 μg supercoiled pBR322 and the topoisomerase I protein were suspendedin reaction buffer (10 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mMbeta-mercaptoethanol, 0.5 mM EDTA). After incubation for 30 minutes at37° C., 0.1% SDS was added to stop the reaction. 0.5 μg/mL proteinase Kwas then added followed by incubation for 30 minutes at 37° C. in orderto degrade the topoisomerase I protein in the solution. The solution wassubsequently subjected to electrophoresis on 1% (w/v) agarose and theDNA was stained with 0.5 μg/mL ethidium bromide. The topoisomerase Iactivity was measured based on observation of an upward shifted DNA bandwith a UV trans illuminator.

Refolding Protocol Example 1

0.5 mL of the above-described denatured protein (RPA70) solution(concentration 0.5 to 1.0 mg/mL) was dripped onto a film comprisingzeolite beta powder spread over and immobilized on the adhesive surfaceof a commercially available tape (Cellotape®), thereby soaking the filmsurface with the solution. After standing for a while in order to ensureadsorption by the protein onto the refolding molding, the solution wasshaken off and the film surface was washed four times with distilledwater in order to completely remove the protein denaturant.

1 mL refolding buffer (prepared from 50 mM HEPES pH 7.5, 0.5 M NaCl, 20mM 2-mercaptoethanol, refolding factor, and nonionic detergent) was thendripped onto the refolding molding and the protein adsorbed on therefolding molding was desorbed and eluted. The refolding molding waswithdrawn and the solution that remained was transferred to a newEppendorf tube and the activity was measured by the gel shift assaydescribed above: activity was present, which confirmed that the RPA70had undergone refolding.

Refolding Protocol Example 2

Denatured RPA70 protein was refolded by entirely the same procedures andprotocol as described above for protocol example 1, with the exceptionthat this refolding protocol example 2 used a two-sided zeolite betafilm molding fabricated using a commercially available two-sidedadhesive tape. Activity was seen in the gel shift assay, which confirmedthat refolding had occurred.

Refolding Protocol Example 3

Denatured RPA70 protein was refolded by entirely the same procedures andprotocol as described above for protocol example 1, with the exceptionthat this refolding protocol example 3 used a molding fabricated bycoating the surface of a commercially available porous alpha-aluminatube (cylindrical, length=5 cm, opening diameter=5 mm) with zeolite betaby in situ synthesis. Activity was seen in the gel shift assay, whichconfirmed that refolding had occurred.

Refolding Protocol Comparative Example 1

Refolding of denatured RPA70 protein used in refolding protocol examples1 to 3 was carried out using finely divided zeolite beta powder. Theprotocol for this is described below. As compared to the use of arefolding molding according to the present invention, no fewer than 3centrifugal separation steps were carried out, requiring the repetitionof supernatant removal and washing each time, and as a consequence thisprotocol based on finely divided zeolite beta powder was verycumbersome.

100 mg finely divided zeolite beta powder was introduced into a 1.5 mLEppendorf tube followed by the addition of 0.5 mL 6 M guanidinehydrochloride·20 mM trisaminomethane trihydrochloride (TrisHCl) pH7.5·0.5 M NaCl·20 mM 2-mercaptoethanol and suspension. To this was thenadded 6 M guanidine hydrochloride·20 mM 2-mercaptoethanol followed byholding for one hour on ice, after which 0.5 mL of the denatured RPA70protein solution (concentration from 0.5 to 1.0 mg/mL) was added. Inorder to ensure adsorption of the protein on the finely divided zeolitebeta powder, this mixture was stirred for 1 hour with a Rotary CultureRCC-100 (Iwaki Glass Co., Ltd.) placed in a cold room.

The finely divided zeolite beta powder was then sedimented bycentrifugation for 5 seconds at 10000×g and the supernatant was removed.In order to completely remove the protein denaturant from the sedimentedfinely divided zeolite beta powder, it was washed 4 times with 1 mL 20mM TrisHCl pH 7.5·20 mM 2-mercaptoethanol (or water) followed bycentrifugation for 5 seconds at 10000×g and discard of the supernatantthereby produced. The remaining finely divided zeolite beta powder wassuspended by the addition thereto of 1 mL refolding buffer (comprising50 mM HEPES pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol, refoldingfactor, and nonionic detergent).

In order to desorb and elute the protein adsorbed on the finely dividedzeolite beta powder, this suspension was again stirred in the cold withthe Rotary Culture RCC-100 (Iwaki Glass Co., Ltd.). The finely dividedzeolite beta powder was thereafter sedimented by centrifugation for 5seconds at 10000×g, and the protein-containing supernatant wastransferred to a new Eppendorf tube and this was used for activitymeasurement by the gel shift assay. Activity was observed, whichconfirmed refolding of the RPA70.

As shown in the preceding examples, activity native to the protein, forexample, DNA binding activity, is rapidly produced by a very simpleprotocol. The refolding molding according to the present invention isuseful as a highly versatile, highly generalizable refolding moldingthat is applicable to a variety of denatured and deactivated proteinsand proteins that have an as yet unformed higher order structure;however, the application thereof is not limited to the proteins shown inthe examples and the refolding molding according to the presentinvention can be applied to any protein.

INDUSTRIAL APPLICABILITY

As has been described hereinabove, the present invention relates, interalia, to a method for activating a function of an inactive protein. Thepresent invention enables the generation by refolding of a nativefunction or activity of protein produced, for example, by an Escherichiacoli expression system, that is inactive due to an as yet unformedhigher order structure, or protein deactivated due to a change inconformation for some reason. This method is useful as a highlyefficient method for refolding inclusion bodies. It can provide anefficient, versatile, and generalizable method that provides a highrefolding rate and that can be applied to a variety of proteins. Thezeolite beta of the function activator used by the present invention isinexpensive and can also be used repeatedly. This method can be appliedto the refolding of a variety of conformation-disordered proteins,including large proteins with a molecular weight in excess of 100,000.For example, by combining the method according to the present inventionwith a protein synthesis process that employs an Escherichia coliexpression system, it becomes possible to elaborate a novel process formanufacturing active protein that produces protein having a controlledhigher order structure and expressing a native function inherent to theprotein.

The present invention additionally provides an efficient, versatile, andgeneralizable refolding kit that has a high refolding rate and that isapplicable to a variety of proteins. The present invention also providesa method for using this refolding kit. The zeolite beta constituting therefolding agent that is an essential component of the kit according tothe present invention is inexpensive and can also be used repeatedly.This refolding kit has the ability to refold a variety ofconformation-disordered proteins, including large proteins with amolecular weight in excess of 100,000. Accordingly, as a furtherdevelopment, through the combination, for example, of the kit accordingto the present invention and its method of use with a process of proteinsynthesis using an Escherichia coli expression system, a novel processand system for manufacturing active protein can be devised that producesprotein having a controlled higher order structure and a native functioninherent to the protein.

The present invention also relates to a function activator and afunction-activating molding for inactive protein. The method using thefunction activator according to the present invention is useful as ahighly efficient method for refolding inclusion bodies. An efficientrefolding agent is provided that is versatile and generalizable and thathas a high refolding rate and that is applicable to a variety ofproteins. The zeolite beta constituting the refolding agent according tothe present invention is inexpensive and can be used repeatedly. Therefolding agent according to the present invention has the ability torefold a variety of conformation-disordered proteins, including largeproteins with a molecular weight in excess of 100,000. Accordingly, as afurther development, through the combination, for example, of therefolding agent according to the present invention and its method of usewith a process of protein synthesis using an Escherichia coli expressionsystem, a novel process and system for manufacturing active protein canbe devised that produces protein having a controlled higher orderstructure and a native function inherent to the protein.

1-8. (canceled)
 9. A protein refolding kit or composition comprisingzeolite with the BEA structure (zeolite beta) as a constituent.
 10. Therefolding kit or composition according to claim 9, further comprising atleast one protein denaturant, pH regulator, an agent that inhibits theformation of protein S—S bridges, surfactant, or refolding factor. 11.The refolding kit or composition according to claim 9, wherein theframework structure of the zeolite beta contains silicon, oxygen, and atleast one element other than silicon and oxygen.
 12. The refolding kitor composition according to claim 9, wherein the framework structure ofthe zeolite beta comprises only silicon and oxygen or only silicon andaluminum and oxygen.
 13. The refolding kit or composition according toclaim 9, wherein the zeolite beta contains an ammonium species.
 14. Therefolding kit or composition according to claim 13, wherein the ammoniumspecies is ammonium ion, an organic amine, and/or an acid amide.
 15. Therefolding kit or composition according to claim 14, wherein the organicamine is a tetraalkylammonium.
 16. The refolding kit or compositionaccording to claim 10, that comprises a protein denaturant that isguanidine hydrochloride.
 17. The refolding kit or composition accordingto claim 10, comprising a pH regulator that is trisaminomethanetrihydrochloride (TrisHCl) and/or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
 18. Therefolding kit or composition according to claim 10, comprising an agentthat inhibits the formation of protein S—S bridges that is2-mercaptoethanol, dithiothreitol, cysteine, and/or thiophenol.
 19. Therefolding kit or composition according to claim 10, comprising at leastone surfactant and refolding factor selected from the group consistingof polyethylene glycol, Ficol170, Ficol1400, polyphosphoric acid, sodiumdodecyl sulfate (SDS), sucrose, glucose, glycerol, inositol,cyclodextrin, amylose, Dextran T-500, Tween 20, Tween 40, Tween 60,NP-40, SB3-14, SB 12, CTAB, and Triton X-100.
 20. The refolding kit orcomposition according to claim 9, comprising a refolding agent,guanidine hydrochloride, TrisHCl, 2-mercaptoethanol, and a solution (therefolding buffer) comprising HEPES, alkali halide, 2-mercaptoethanol,refolding factor, and surfactant, or that comprises the refoldingbuffer, the refolding agent, guanidine hydrochloride, TrisHCl,2-mercapethanol, and alkali halide. 21-42. (canceled)
 43. The proteinrefolding kit or composition of claim 9, wherein the zeolite containsammonium ion, an organic ammonium ion, or urea.
 44. The proteinrefolding kit or composition of claim 9, wherein the zeolite containsammonium ion.
 45. The protein refolding kit or composition of claim 9,wherein the zeolite contains urea.
 46. The protein refolding kit orcomposition of claim 9, wherein the zeolite an organic ammonium ion thatis a mono-, di-, tri- and/or tetra-alkylammonium ion, wherein said alkylgroup is at least one of methyl, ethyl, propyl, or butyl.
 47. Theprotein refolding kit or composition of claim 9, further comprising atleast one protein.